Rnai agents, compositions and methods of use thereof for treating transthyretin (ttr) associated diseases

ABSTRACT

The present invention provides RNAi agents, e.g., double stranded RNAi agents, that target the transthyretin (TTR) gene and methods of using such RNAi agents for treating or preventing TTR-associated diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/738,014, filed Jan. 9, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/188,317, filed on Jun. 21, 2016, now U.S. Pat.No. 10,570,391, issued on Feb. 25, 2020, which is a continuation of U.S.patent application Ser. No. 14/358,972, filed on May 16, 2014, now U.S.Pat. No. 9,399,775, issued on Jul. 26, 2016, which is a 35 U.S.C. § 371national stage filing of International Application No.PCT/US2012/065691, filed on Nov. 16, 2012, which claims priority to U.S.Provisional Application No. 61/561,710, filed on Nov. 18, 2011, U.S.Provisional Application No. 61/615,618, filed on Mar. 26, 2012, and U.S.Provisional Application No. 61/680,098, filed on Aug. 6, 2012. Theentire contents of each of the foregoing applications are herebyincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 8, 2021, isnamed 121301_00106_SL.txt and is 541,682 bytes in size.

BACKGROUND OF THE INVENTION

Transthyretin (TTR) (also known as prealbumin) is found in serum andcerebrospinal fluid (CSF). TTR transports retinol-binding protein (RBP)and thyroxine (T4) and also acts as a carrier of retinol (vitamin A)through its association with RBP in the blood and the CSF. Transthyretinis named for its transport of thyroxine and retinol. TTR also functionsas a protease and can cleave proteins including apoA-I (the major HDLapolipoprotein), amyloid β-peptide, and neuropeptide Y. See Liz, M. A.et al. (2010) IUBMB Life, 62(6):429-435.

TTR is a tetramer of four identical 127-amino acid subunits (monomers)that are rich in beta sheet structure. Each monomer has two 4-strandedbeta sheets and the shape of a prolate ellipsoid. Antiparallelbeta-sheet interactions link monomers into dimers. A short loop fromeach monomer forms the main dimer-dimer interaction. These two pairs ofloops separate the opposed, convex beta-sheets of the dimers to form aninternal channel.

The liver is the major site of TTR expression. Other significant sitesof expression include the choroid plexus, retina (particularly theretinal pigment epithelium) and pancreas. Transthyretin is one of atleast 27 distinct types of proteins that is a precursor protein in theformation of amyloid fibrils. See Guan, J. et al. (Nov. 4, 2011) Currentperspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol,doi:10.1152/ajpheart.00815.2011. Extracellular deposition of amyloidfibrils in organs and tissues is the hallmark of amyloidosis. Amyloidfibrils are composed of misfolded protein aggregates, which may resultfrom either excess production of or specific mutations in precursorproteins. The amyloidogenic potential of TTR may be related to itsextensive beta sheet structure; X-ray crystallographic studies indicatethat certain amyloidogenic mutations destabilize the tetramericstructure of the protein. See, e.g., Saraiva M. J. M. (2002) ExpertReviews in Molecular Medicine, 4(12):1-11.

Amyloidosis is a general term for the group of amyloid diseases that arecharacterized by amyloid deposits. Amyloid diseases are classified basedon their precursor protein; for example, the name starts with “A” foramyloid and is followed by an abbreviation of the precursor protein,e.g., ATTR for amyloidogenic transthyretin. Ibid.

There are numerous TTR-associated diseases, most of which are amyloiddiseases. Normal-sequence TTR is associated with cardiac amyloidosis inpeople who are elderly and is termed senile systemic amyloidosis (SSA)(also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis).SSA often is accompanied by microscopic deposits in many other organs.TTR amyloidosis manifests in various forms. When the peripheral nervoussystem is affected more prominently, the disease is termed familialamyloidotic polyneuropathy (FAP). When the heart is primarily involvedbut the nervous system is not, the disease is called familialamyloidotic cardiomyopathy (FAC). A third major type of TTR amyloidosisis leptomeningeal amyloidosis, also known as leptomeningeal ormeningocerebrovascular amyloidosis, central nervous system (CNS)amyloidosis, or amyloidosis VII form. Mutations in TTR may also causeamyloidotic vitreous opacities, carpal tunnel syndrome, and euthyroidhyperthyroxinemia, which is a non-amyloidotic disease thought to besecondary to an increased association of thyroxine with TTR due to amutant TTR molecule with increased affinity for thyroxine. See, e.g.,Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.

Abnormal amyloidogenic proteins may be either inherited or acquiredthrough somatic mutations. Guan, J. et al. (Nov. 4, 2011) Currentperspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol,doi:10.1152/ajpheart.00815.2011. Transthyretin associated ATTR is themost frequent form of hereditary systemic amyloidosis. Lobato, L. (2003)J. Nephrol., 16:438-442. TTR mutations accelerate the process of TTRamyloid formation and are the most important risk factor for thedevelopment of ATTR. More than 85 amyloidogenic TTR variants are knownto cause systemic familial amyloidosis. TTR mutations usually give riseto systemic amyloid deposition, with particular involvement of theperipheral nervous system, although some mutations are associated withcardiomyopathy or vitreous opacities. Ibid.

The V30M mutation is the most prevalent TTR mutation. See, e.g., Lobato,L. (2003) J Nephrol, 16:438-442. The V122I mutation is carried by 3.9%of the African American population and is the most common cause of FAC.Jacobson, D. R. et al. (1997) N. Engl. J. Med. 336 (7): 466-73. It isestimated that SSA affects more than 25% of the population over age 80.Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87 (7):2843-5.

Accordingly, there is a need in the art for effective treatments forTTR-associated diseases.

SUMMARY OF THE INVENTION

The present invention provides RNAi agents, e.g., double stranded RNAiagents, targeting the Transthyretin (TTR) gene. The present inventionalso provides methods of inhibiting expression of TTR and methods oftreating or preventing a TTR-associated disease in a subject using theRNAi agents, e.g. double stranded RNAi agents, of the invention. Thepresent invention is based, at least in part, on the discovery that RNAiagents that comprise particular chemical modifications show a superiorability to inhibit expression of TTR. Agents including a certain patternof chemical modifications (e.g., an alternating pattern) and a ligandare shown herein to be effective in silencing the activity of the TTRgene. Furthermore, agents including one or more motifs of threeidentical modifications on three consecutive nucleotides, including onesuch motif at or near the cleavage site of the agents, show surprisinglyenhanced TTR gene silencing activity. When a single such chemical motifis present in the agent, it is preferred to be at or near the cleavageregion for enhancing of the gene silencing activity. Cleavage region isthe region surrounding the cleavage site, i.e., the site on the targetmRNA at which cleavage occurs.

Accordingly, in one aspect, the present invention features RNAi agents,e.g., double stranded RNAi agents, for inhibiting expression of atransthyretin (TTR). The double stranded RNAi agent includes a sensestrand complementary to an antisense strand. The antisense strandincludes a region complementary to a part of an mRNA encodingtransthyretin. Each strand has 14 to 30 nucleotides, and the doublestranded RNAi agent is represented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)-N_(a)′-n_(d)′5′   (III).

In Formula III, i, j, k, and l are each independently 0 or 1; p, p′, q,and q′ are each independently 0-6; each N_(a) and N_(a)′ independentlyrepresents an oligonucleotide sequence including 0-25 nucleotides whichare either modified or unmodified or combinations thereof, each sequenceincluding at least two differently modified nucleotides; each N_(b) andN_(b)′ independently represents an oligonucleotide sequence including0-10 nucleotides which are either modified or unmodified or combinationsthereof, each n_(p), n_(p)′, n_(q), and n_(q)′ independently representsan overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ eachindependently represents one motif of three identical modifications onthree consecutive nucleotides; modifications on N_(b) differ from themodification on Y and modifications on N_(b)′ differ from themodification on Y′. In some embodiments, the sense strand is conjugatedto at least one ligand, e.g., at least one ligand, e.g., at least oneligand attached to the 3′ end of the sense strand. In other embodiments,the ligand may be conjugated to the antisense strand.

In some embodiments, i is 1; j is 1; or both i and j are 1.

In some embodiments, k is 1; l is 1; or both k and l are 1.

In some embodiments, i is 0; j is 1.

In some embodiments, i is 1, j is 0.

In some embodiments, k is 0; l is 1.

In some embodiments, k is 1; l is 0.

In some embodiments, XXX is complementary to X′X′X′, YYY iscomplementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In some embodiments, the YYY motif occurs at or near the cleavage siteof the sense strand.

In some embodiments, the Y′Y′Y′ motif occurs at the 11, 12 and 13positions of the antisense strand from the 5′-end.

In some embodiments, the Y′ is 2′-O-methyl.

In some embodiments, the Y′ is 2′-fluoro.

In some embodiments, formula (III) is represented as formula (IIIa):

sense: 5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

antisense: 3′n _(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a) ′n _(q)′5′  (IIIa).

In formula IIIa, each N_(b) and N_(b)′ independently represents anoligonucleotide sequence including 1-5 modified nucleotides.

In some embodiments, formula (III) is represented as formula (IIIb):

sense: 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′

antisense: 3′n _(p)′-N′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n _(q)′5′   (IIIb).

In formula IIIb each N_(b) and N_(b)′ independently represents anoligonucleotide sequence including 1-5 modified nucleotides.

In some embodiments, formula (III) is represented as formula (IIIc):

sense: 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

antisense: 3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′5′   (IIc).

In formula IIIc, each N_(b) and —N_(b)′ independently represents anoligonucleotide sequence including 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequenceincluding 2-10 modified nucleotides.

In many embodiments, the duplex region is 15-30 nucleotide pairs inlength. In some embodiments, the duplex region is 17-23 nucleotide pairsin length, 17-25 nucleotide pairs in length, 23-27 nucleotide pairs inlength, 19-21 nucleotide pairs in length, or 21-23 nucleotide pairs inlength.

In certain embodiments, each strand has 15-30 nucleotides.

In some embodiments, the modifications on the nucleotides are selectedfrom the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl,and combinations thereof. In some preferred embodiments, themodifications on the nucleotides are 2′-O-methyl or 2′-fluoro.

In some embodiments, the ligand is one or more N-acetylgalactosamine(GalNAc) derivatives attached through a bivalent or trivalent branchedlinker. In particular embodiments the ligand is

In some embodiments, the ligand is attached to the 3′ end of the sensestrand.

In some embodiments, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is 0 or S.

In some embodiments, the RNAi agent is conjugated to the ligand as shownin the following schematic

In some embodiments, the RNAi agent further includes at least onephosphorothioate or methylphosphonate internucleotide linkage. In someembodiments, the phosphorothioate or methylphosphonate internucleotidelinkage is at the 3′-terminal of one strand. In some embodiments, thestrand is the antisense strand. In other embodiments, the strand is thesense strand.

In certain embodiments, the base pair at the 1 position of the 5′-end ofthe duplex is an AU base pair.

In some embodiments, the Y nucleotides contain a 2′-fluoro modification.

In some embodiments, the Y′ nucleotides contain a 2′-O-methylmodification.

In some embodiments, p′>0. In some such embodiments, each n iscomplementary to the target mRNA. In other such embodiments, each n isnon-complementary to the target mRNA. In some embodiments, p, p′, q andq′ are 1-6. In some preferred embodiments, p′=1 or 2. In some preferredembodiments, p′=2. In some such embodiments, q′=0, p=0, q=0, and p′overhang nucleotides are complementary to the target mRNA. In other suchembodiments, q′=0, p=0, q=0, and p′ overhang nucleotides arenon-complementary to the target mRNA.

In some embodiments, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides.

In certain embodiments, linkages between n_(p)′ include phosphorothioatelinkages. In some such embodiments, the linkages between n_(p)′ arephosphorothioate linkages.

In some embodiments, the RNAi agent is selected from the group of agentslisted in Table 1.

In preferred embodiments, the RNAi agent is selected from the groupconsisting of AD-51544, AD-51545, AD-51546, and AD-51547.

In an even more preferred embodiment, the RNAi agent is AD-51547 havingthe following structure:

sense: (SEQ ID NO: 2) 5′- UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′antisense: (SEQ ID NO: 3) 5′- uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′wherein lowercase nucleotides (a, u, g, c) indicate 2′-O-methylnucleotides; Nf (e.g., Af) indicates a 2′-fluoro nucleotide; s indicatesa phosphothiorate linkage; L96 indicates a GalNAc₃ ligand.

In another aspect, the present invention features a cell containing theRNAi agent for inhibiting expression of TTR.

In a further aspect, the present invention features a pharmaceuticalcomposition comprising an RNAi agent for inhibiting expression of TTR.In some embodiments, the pharmaceutical composition is a solutioncomprising the RNAi agent. In some embodiments, the solution comprisingthe RNAi agent is an unbuffered solution, e.g., saline solution orwater. In other embodiments, the solution is a buffered solution, e.g.,a solution of phosphate buffered saline (PBS). In other embodiments, thepharmaceutical composition is a liposome or a lipid formulation. In someembodiments, the lipid formulation comprises a XTC or MC3.

In yet another aspect, the present invention features methods ofinhibiting expression of transthyretin (TTR) in a cell. The methodsinclude contacting a cell with an RNAi agent, e.g., a double strandedRNAi agent, in an amount effective to inhibit expression of TTR in thecell, thereby inhibiting expression of TTR in the cell.

In some embodiments, the expression of TTR is inhibited by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or at least about 90%.

In other embodiments, the cell is contacted in vitro with the RNAiagent. In other embodiments, the cell is present within a subject. Inpreferred embodiments, the subject is a human.

In further embodiments, the subject is a subject suffering from aTTR-associated disease and the effective amount is a therapeuticallyeffective amount. In other embodiments, the subject is a subject at riskfor developing a TTR-associated disease and the effective amount is aprophylactically effective amount. In some embodiments, a subject atrisk for developing a TTR-associated disease is a subject who carries aTTR gene mutation that is associated with the development of aTTR-associated disease.

In certain embodiments, the TTR-associated disease is selected from thegroup consisting of senile systemic amyloidosis (SSA), systemic familialamyloidosis, familial amyloidotic polyneuropathy (FAP), familialamyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System(CNS) amyloidosis, and hyperthyroxinemia.

In some embodiments, the subject has a TTR-associated amyloidosis andthe method reduces an amyloid TTR deposit in the subject.

In other embodiments, the RNAi agent is administered to the subject byan administration means selected from the group consisting ofsubcutaneous, intravenous, intramuscular, intrabronchial, intrapleural,intraperitoneal, intraarterial, lymphatic, cerebrospinal, and anycombinations thereof. In certain embodiments, the RNAi agent isadministered to the subject via subcutaneous or intravenousadministration. In preferred embodiments, the RNAi agent is administeredto the subject via subcutaneous administration. In some suchembodiments, the subcutaneous administration includes administration viaa subcutaneous pump or subcutaneous depot.

In certain embodiments, the RNAi agent is administered to the subjectsuch that the RNAi agent is delivered to a specific site within thesubject. In some embodiments, the site is selected from the groupconsisting of liver, choroid plexus, retina, and pancreas. In preferredembodiments, the site is the liver. In some embodiments, the delivery ofthe RNAi agent is mediated by asialoglycoprotein receptor (ASGP-R)present in hepatocytes.

In some embodiments, the RNAi agent is administered at a dose of betweenabout 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg toabout 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, betweenabout 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg toabout 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg,between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg toabout 50 mg/kg.

In some embodiments, the RNAi agent is administered at a dose of about0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg,about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg,about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg,about 49 mg/kg or about 50 mg/kg.

In some embodiments, the RNAi agent is administered in two or moredoses. In particular embodiments, the RNAi agent is administered atintervals selected from the group consisting of once every about 2hours, once every about 3 hours, once every about 4 hours, once everyabout 6 hours, once every about 8 hours, once every about 12 hours, onceevery about 24 hours, once every about 48 hours, once every about 72hours, once every about 96 hours, once every about 120 hours, once everyabout 144 hours, once every about 168 hours, once every about 240 hours,once every about 336 hours, once every about 504 hours, once every about672 hours and once every about 720 hours.

In other embodiments, the method further includes assessing the level ofTTR mRNA expression or TTR protein expression in a sample derived fromthe subject.

In preferred embodiments, administering the RNAi agent does not resultin an inflammatory response in the subject as assessed based on thelevel of a cytokine or chemokine selected from the group consisting ofG-CSF, IFN-γ, IL-10, IL-12 (p70), IL1β, IL-Ira, IL-6, IL-8, IP-10,MCP-1, MIP-1α, MIP-1β, TNFα, and any combinations thereof, in a samplefrom the subject.

In some embodiments, the RNAi agent is administered using apharmaceutical composition

In preferred embodiments, the RNAi agent is administered in a solution.In some such embodiments, the siRNA is administered in an unbufferedsolution. In one embodiment, the siRNA is administered in water. Inother embodiments, the siRNA is administered with a buffer solution,such as an acetate buffer, a citrate buffer, a prolamine buffer, acarbonate buffer, or a phosphate buffer or any combination thereof. Insome embodiments, the buffer solution is phosphate buffered saline(PBS).

In another embodiment, the pharmaceutical composition is a liposome or alipid formulation comprising SNALP or XTC. In one embodiment, the lipidformulation comprises an MC3.

In another aspect, the invention provides methods of treating orpreventing a TTR-associated disease in a subject. The methods includeadministering to the subject a therapeutically effective amount orprophylactically effective amount of an RNAi agent, e.g., a doublestranded RNAi agent, thereby treating or preventing the TTR-associateddisease in the subject.

In some embodiments, TTR expression in a sample derived from the subjectis inhibited by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60% or atleast about 70% at least about 80%, or at least about 90%.

In some embodiments, the subject is a human.

In some embodiments, the subject is a subject suffering from aTTR-associated disease. In other embodiments, the subject is a subjectat risk for developing a TTR-associated disease.

In some embodiments, the subject is a subject who carries s a TTR genemutation that is associated with the development of a TTR-associateddisease.

In certain embodiments, the TTR-associated disease is selected from thegroup consisting of senile systemic amyloidosis (SSA), systemic familialamyloidosis, familial amyloidotic polyneuropathy (FAP), familialamyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System(CNS) amyloidosis, and hyperthyroxinemia.

In some embodiments, the subject has a TTR-associated amyloidosis andthe method reduces an amyloid TTR deposit in the subject.

In some embodiments, the RNAi agent is administered to the subject by anadministration means selected from the group consisting of subcutaneous,intravenous, intramuscular, intrabronchial, intrapleural,intraperitoneal, intraarterial, lymphatic, cerebrospinal, and anycombinations thereof. In certain embodiments, the RNAi agent isadministered to the subject via subcutaneous or intravenousadministration. In preferred embodiments, the RNAi agent is administeredto the subject via subcutaneous administration. In some suchembodiments, the subcutaneous administration includes administration viaa subcutaneous pump or subcutaneous depot.

In certain embodiments, the RNAi agent is administered to the subjectsuch that the RNAi agent is delivered to a specific site within thesubject. In some such embodiments, the site is selected from the groupconsisting of liver, choroid plexus, retina, and pancreas. In preferredembodiments, the site is the liver. In some embodiments, the delivery ofthe RNAi agent is mediated by asialoglycoprotein receptor (ASGP-R)present in hepatocytes.

In some embodiments, the RNAi agent is administered at a dose of betweenabout 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg toabout 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, betweenabout 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg toabout 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg,between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg toabout 50 mg/kg.

In some embodiments, the RNAi agent is administered at a dose of about0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg,about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg,about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg,about 49 mg/kg or about 50 mg/kg.

In some embodiments, the RNAi agent is administered in two or moredoses. In particular embodiments, the RNAi agent is administered atintervals selected from the group consisting of once every about 2hours, once every about 3 hours, once every about 4 hours, once everyabout 6 hours, once every about 8 hours, once every about 12 hours, onceevery about 24 hours, once every about 48 hours, once every about 72hours, once every about 96 hours, once every about 120 hours, once everyabout 144 hours, once every about 168 hours, once every about 240 hours,once every about 336 hours, once every about 504 hours, once every about672 hours and once every about 720 hours.

In other embodiments, the method further includes assessing the level ofTTR mRNA expression or TTR protein expression in a sample derived fromthe subject.

In preferred embodiments, administering the RNAi agent does not resultin an inflammatory response in the subject as assessed based on thelevel of a cytokine or chemokine selected from the group consisting ofG-CSF, IFN-γ, IL-10, IL-12 (p70), IL1β, IL-Ira, IL-6, IL-8, IP-10,MCP-1, MIP-1α, MIP-1β, TNFα, and any combinations thereof, in a samplefrom the subject.

In some embodiments, the RNAi agent is administered using apharmaceutical composition, e.g., a liposome.

In some embodiments, the RNAi agent is administered in a solution. Insome such embodiments, the siRNA is administered in an unbufferedsolution. In one embodiment, the siRNA is administered in saline orwater. In other embodiments, the siRNA is administered with a buffersolution, such as an acetate buffer, a citrate buffer, a prolaminebuffer, a carbonate buffer, or a phosphate buffer or any combinationthereof. In some embodiments, the buffer solution is phosphate bufferedsaline (PBS).

In another aspect, the present invention provides a method of inhibitingexpression of transthyretin (TTR) in a cell, including contacting a cellwith an RNAi agent, e.g., a double stranded RNAi agent, in an amounteffective to inhibit expression of TTR in the cell. In one aspect, thedouble stranded RNAi agent is selected from the group of agents listedin Table 1, thereby inhibiting expression of transthyretin (TTR) in thecell.

In another aspect, the present invention provides a method of inhibitingexpression of transthyretin (TTR) in a cell, including contacting a cellwith an RNAi agent, e.g., a double stranded RNAi agent, in an amounteffective to inhibit expression of TTR in the cell. In one aspect, thedouble stranded RNAi agent is selected from the group consisting ofAD-51544, AD-51545, AD-51546, and AD-51547, thereby inhibitingexpression of transthyretin (TTR) in the cell.

In a further aspect, the present invention provides a method of treatingor preventing a TTR-associated disease in a subject, includingadministering to the subject a therapeutically effective amount or aprophylactically effective amount of an RNAi agent, e.g., a doublestranded RNAi agent. In one aspect, the double stranded RNAi agent isselected from the group of agents listed in Table 1, thereby treating orpreventing a TTR-associated disease in the subject.

In yet another aspect, the present invention provides a method oftreating or preventing a TTR-associated disease in a subject, includingadministering to the subject a therapeutically effective amount or aprophylactically effective amount of an RNAi agent, e.g., a doublestranded RNAi agent. In one aspect, the double stranded RNAi agent isselected from the group consisting of AD-51544, AD-51545, AD-51546, andAD-51547, thereby treating or preventing a TTR-associated disease in thesubject.

In further aspects, the invention provides kits for performing themethods of the invention. In one aspect, the invention provides a kitfor performing a method of inhibiting expression of transthyretin (TTR)in a cell comprising contacting a cell with an RNAi agent, e.g., adouble stranded RNAi agent, in an amount effective to inhibit expressionof said TTR in said cell, thereby inhibiting the expression of TTR inthe cell. The kit comprises an RNAi agent and instructions for use and,optionally, means for administering the RNAi agent to the subject.

The present invention is further illustrated by the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting that administering to mice a singlesubcutaneous dose of a GalNAc-conjugated RNAi agent targeting TTRresulted in dose-dependent suppression of TTR mRNA.

FIG. 2 is a graph depicting that administering to mice a singlesubcutaneous dose of 7.5 mg/kg or 30 mg/kg of a GalNAc conjugated RNAiagent targeting TTR resulted in long lasting suppression of TTR mRNA.

FIG. 3 depicts the human TTR mRNA sequence.

FIG. 4 is a graph depicting improved silencing activity of RNAi agentsmodified relative to the parent AD-45163.

FIG. 5 is a graph depicting improved silencing activity of RNAi agentsmodified relative to the parent AD-45165.

FIG. 6 is a graph depicting improved free uptake silencing following 4hour incubation with RNAi agents modified relative to the parentAD-45163.

FIG. 7 is a graph depicting improved free uptake silencing following 24hour incubation with RNAi agents modified relative to the parentAD-45163.

FIG. 8 is a graph depicting improved free uptake silencing following 4hour incubation with RNAi agents modified relative to the parentAD-45165.

FIG. 9 is a graph depicting improved free uptake silencing following 24hour incubation with RNAi agents modified relative to the parentAD-45165.

FIG. 10A is a graph depicting silencing of TTR mRNA in transgenic micethat express hTTR V30M following administration of a single subcutaneousdose of RNAi agents AD-51544, AD-51545, or AD-45163.

FIG. 10B is a graph depicting silencing of TTR mRNA in transgenic micethat express hTTR V30M following administration of a single subcutaneousdose of RNAi agents AD-51546, AD-51547, or AD-45165.

FIG. 11 is a graph depicting TTR protein suppression in transgenic micethat express hTTR V30M following administration of a single subcutaneousdose of 5 mg/kg or 1 mg/kg of RNAi agents AD-51544, AD-51545, orAD-45163.

FIG. 12 is a graph depicting TTR protein suppression in transgenic micethat express hTTR V30M following administration of a single subcutaneousdose of 5 mg/kg or 1 mg/kg of RNAi agents AD-51546, AD-51547, orAD-45165.

FIG. 13 depicts the protocol for post-dose blood draws in monkeys thatreceived 5×5 mg/kg RNAi agent (top line) or 1×25 mg/kg RNAi agent(bottom line).

FIG. 14A is a graph depicting suppression of TTR protein in non-humanprimates following subcutaneous administration of five 5 mg/kg doses ofAD-45163, AD-51544, AD-51545, AD-51546, or AD-51547.

FIG. 14B is a graph depicting suppression of TTR protein in non-humanprimates following subcutaneous administration of a single 25 mg/kg doseof AD-45163, AD-51544, AD-51545, AD-51546, or AD-51547.

FIG. 15 is a graph depicting suppression of TTR protein in non-humanprimates following subcutaneous administration of AD-51547 at 2.5 mg/kg(white squares), 5 mg/kg (black squares) or 10 mg/kg (patterned squares)per dose, or administration of PBS as a negative control (gray squares).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides RNAi agents, e.g., double stranded RNAiagents, and compositions targeting the Transthyretin (TTR) gene. Thepresent invention also provides methods of inhibiting expression of TTRand methods of treating or preventing a TTR-associated disease in asubject using the RNAi agents, e.g., double stranded RNAi agents, of theinvention. The present invention is based, at least in part, on thediscovery that RNAi agents that comprise particular chemicalmodifications show a superior ability to inhibit expression of TTR.Agents including a certain pattern of chemical modifications (e.g., analternating pattern) and a ligand are shown herein to be effective insilencing the activity of the TTR gene. Furthermore, agents includingone or more motifs of three identical modifications on three consecutivenucleotides, including one such motif at or near the cleavage site ofthe agents, show surprisingly enhanced TTR gene silencing activity. Whena single such chemical motif is present in the agent, it is preferred tobe at or near the cleavage region for enhancing of the gene silencingactivity. Cleavage region is the region surrounding the cleavage site,i.e., the site on the target mRNA at which cleavage occurs.

I. Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, a “transthyretin” (“TTR”) refers to the well known geneand protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA.TTR functions as a transporter of retinol-binding protein (RBP),thyroxine (T4) and retinol, and it also acts as a protease. The liversecretes TTR into the blood, and the choroid plexus secretes TTR intothe cerebrospinal fluid. TTR is also expressed in the pancreas and theretinal pigment epithelium. The greatest clinical relevance of TTR isthat both normal and mutant TTR protein can form amyloid fibrils thataggregate into extracellular deposits, causing amyloidosis. See, e.g.,Saraiva M. J. M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11for a review. The molecular cloning and nucleotide sequence of rattransthyretin, as well as the distribution of mRNA expression, wasdescribed by Dickson, P. W. et al. (1985) J. Biol. Chem.260(13)8214-8219. The X-ray crystal structure of human TTR was describedin Blake, C. C. et al. (1974) J Mol Biol 88, 1-12. The sequence of ahuman TTR mRNA transcript can be found at National Center forBiotechnology Information (NCBI) RefSeq accession number NM_000371. Thesequence of mouse TTR mRNA can be found at RefSeq accession numberNM_013697.2, and the sequence of rat TTR mRNA can be found at RefSeqaccession number NM_012681.1

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a TTR gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dt” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine, 2′-deoxythymidine or thymidine. However, it will beunderstood that the term “ribonucleotide” or “nucleotide” or“deoxyribonucleotide” can also refer to a modified nucleotide, asfurther detailed below, or a surrogate replacement moiety. The skilledperson is well aware that guanine, cytosine, adenine, and uracil may bereplaced by other moieties without substantially altering the basepairing properties of an oligonucleotide comprising a nucleotide bearingsuch replacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

A “double stranded RNAi agent,” double-stranded RNA (dsRNA) molecule,also referred to as “dsRNA agent,” “dsRNA”, “siRNA”, “iRNA agent,” asused interchangeably herein, refers to a complex of ribonucleic acidmolecules, having a duplex structure comprising two anti-parallel andsubstantially complementary, as defined below, nucleic acid strands. Ingeneral, the majority of nucleotides of each strand are ribonucleotides,but as described in detail herein, each or both strands can also includeone or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or amodified nucleotide. In addition, as used in this specification, an“RNAi agent” may include ribonucleotides with chemical modifications; anRNAi agent may include substantial modifications at multiplenucleotides. Such modifications may include all types of modificationsdisclosed herein or known in the art. Any such modifications, as used ina siRNA type molecule, are encompassed by “RNAi agent” for the purposesof this specification and claims.

In another embodiment, the RNAi agent may be a single-stranded siRNAthat is introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150: 883-894.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, an RNAi agent may compriseone or more nucleotide overhangs. The term “siRNA” is also used hereinto refer to an RNAi agent as described above.

In another aspect, the agent is a single-stranded antisense RNAmolecule. An antisense RNA molecule is complementary to a sequencewithin the target mRNA. Antisense RNA can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. The antisense RNA molecule may have about 15-30nucleotides that are complementary to the target mRNA. For example, theantisense RNA molecule may have a sequence of at least 15, 16, 17, 18,19, 20 or more contiguous nucleotides from one of the antisensesequences of Table 1.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of anRNAi agent when a 3′-end of one strand of the RNAi agent extends beyondthe 5′-end of the other strand, or vice versa. “Blunt” or “blunt end”means that there are no unpaired nucleotides at that end of the doublestranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAiagent is a dsRNA that is double-stranded over its entire length, i.e.,no nucleotide overhang at either end of the molecule. The RNAi agents ofthe invention include RNAi agents with nucleotide overhangs at one end(i.e., agents with one overhang and one blunt end) or with nucleotideoverhangs at both ends.

The term “antisense strand” refers to the strand of a double strandedRNAi agent which includes a region that is substantially complementaryto a target sequence (e.g., a human TTR mRNA). As used herein, the term“region complementary to part of an mRNA encoding transthyretin” refersto a region on the antisense strand that is substantially complementaryto part of a TTR mRNA sequence. Where the region of complementarity isnot fully complementary to the target sequence, the mismatches are mosttolerated in the terminal regions and, if present, are generally in aterminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides ofthe 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Sequences can be “fully complementary” with respect to each when thereis base-pairing of the nucleotides of the first nucleotide sequence withthe nucleotides of the second nucleotide sequence over the entire lengthof the first and second nucleotide sequences. However, where a firstsequence is referred to as “substantially complementary” with respect toa second sequence herein, the two sequences can be fully complementary,or they may form one or more, but generally not more than 4, 3 or 2mismatched base pairs upon hybridization, while retaining the ability tohybridize under the conditions most relevant to their ultimateapplication. However, where two oligonucleotides are designed to form,upon hybridization, one or more single stranded overhangs, suchoverhangs shall not be regarded as mismatches with regard to thedetermination of complementarity. For example, a dsRNA comprising oneoligonucleotide 21 nucleotides in length and another oligonucleotide 23nucleotides in length, wherein the longer oligonucleotide comprises asequence of 21 nucleotides that is fully complementary to the shorteroligonucleotide, may yet be referred to as “fully complementary” for thepurposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding TTR) including a 5′ UTR, an openreading frame (ORF), or a 3′ UTR. For example, a polynucleotide iscomplementary to at least a part of a TTR mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding TTR.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a TTR,” as used herein, includesinhibition of expression of any TTR gene (such as, e.g., a mouse TTRgene, a rat TTR gene, a monkey TTR gene, or a human TTR gene) as well asvariants or mutants of a TTR gene. Thus, the TTR gene may be a wild-typeTTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise tosystemic amyloid deposition), or a transgenic TTR gene in the context ofa genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a TTR gene” includes any level of inhibitionof a TTR gene, e.g., at least partial suppression of the expression of aTTR gene, such as an inhibition of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%. at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99%.

The expression of a TTR gene may be assessed based on the level of anyvariable associated with TTR gene expression, e.g., TTR mRNA level, TTRprotein level, retinol binding protein level, vitamin A level, or thenumber or extent of amyloid deposits. Inhibition may be assessed by adecrease in an absolute or relative level of one or more of thesevariables compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

The phrase “contacting a cell with an RNAi agent,” as used herein,includes contacting a cell by any possible means. Contacting a cell withan RNAi agent, e.g., a double stranded RNAi agent, includes contacting acell in vitro with the RNAi agent or contacting a cell in vivo with theRNAi agent. The contacting may be done directly or indirectly. Thus, forexample, the RNAi agent may be put into physical contact with the cellby the individual performing the method, or alternatively, the RNAiagent may be put into a situation that will permit or cause it tosubsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., a GalNAc₃ ligand, that directs the RNAi agent to a site ofinterest, e.g., the liver. Combinations of in vitro and in vivo methodsof contacting are also possible. In connection with the methods of theinvention, a cell might also be contacted in vitro with an RNAi agentand subsequently transplanted into a subject.

A “patient” or “subject,” as used herein, is intended to include eithera human or non-human animal, preferably a mammal, e.g., a monkey. Mostpreferably, the subject or patient is a human.

A “TTR-associated disease,” as used herein, is intended to include anydisease associated with the TTR gene or protein. Such a disease may becaused, for example, by excess production of the TTR protein, by TTRgene mutations, by abnormal cleavage of the TTR protein, by abnormalinteractions between TTR and other proteins or other endogenous orexogenous substances. A “TTR-associated disease” includes any type ofTTR amyloidosis (ATTR) wherein TTR plays a role in the formation ofabnormal extracellular aggregates or amyloid deposits. TTR-associateddiseases include senile systemic amyloidosis (SSA), systemic familialamyloidosis, familial amyloidotic polyneuropathy (FAP), familialamyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System(CNS) amyloidosis, amyloidotic vitreous opacities, carpal tunnelsyndrome, and hyperthyroxinemia. Symptoms of TTR amyloidosis includesensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs),autonomic neuropathy (e.g., gastrointestinal dysfunction, such asgastric ulcer, or orthostatic hypotension), motor neuropathy, seizures,dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomicinsufficiency, cardiomyopathy, vitreous opacities, renal insufficiency,nephropathy, substantially reduced mBMI (modified Body Mass Index),cranial nerve dysfunction, and corneal lattice dystrophy.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a patientfor treating a TTR associated disease, is sufficient to effect treatmentof the disease (e.g., by diminishing, ameliorating or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, stage ofpathological processes mediated by TTR expression, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjectwho does not yet experience or display symptoms of a TTR-associateddisease, but who may be predisposed to the disease, is sufficient toprevent or ameliorate the disease or one or more symptoms of thedisease. Symptoms that may be ameliorated include sensory neuropathy(e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy(e.g., gastrointestinal dysfunction, such as gastric ulcer, ororthostatic hypotension), motor neuropathy, seizures, dementia,myelopathy, polyneuropathy, carpal tunnel syndrome, autonomicinsufficiency, cardiomyopathy, vitreous opacities, renal insufficiency,nephropathy, substantially reduced mBMI (modified Body Mass Index),cranial nerve dysfunction, and corneal lattice dystrophy. Amelioratingthe disease includes slowing the course of the disease or reducing theseverity of later-developing disease. The “prophylactically effectiveamount” may vary depending on the RNAi agent, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically-effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. RNAi gents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes), the retina or parts of the retina (e.g., retinal pigmentepithelium), the central nervous system or parts of the central nervoussystem (e.g., ventricles or choroid plexus), or the pancreas or certaincells or parts of the pancreas. In some embodiments, a “sample derivedfrom a subject” refers to cerebrospinal fluid obtained from the subject.In preferred embodiments, a “sample derived from a subject” refers toblood or plasma drawn from the subject. In further embodiments, a“sample derived from a subject” refers to liver tissue (or subcomponentsthereof) or retinal tissue (or subcomponents thereof) derived from thesubject.

II. RNAi Agents

The present invention provides RNAi agents with superior gene silencingactivity. It is shown herein and in Provisional Application No.61/561,710 (to which the present application claims priority) that asuperior result may be obtained by introducing one or more motifs ofthree identical modifications on three consecutive nucleotides into asense strand and/or antisense strand of a RNAi agent, particularly at ornear the cleavage site. The sense strand and antisense strand of theRNAi agent may otherwise be completely modified. The introduction ofthese motifs interrupts the modification pattern, if present, of thesense and/or antisense strand. The RNAi agent also optionally conjugateswith a GalNAc derivative ligand, for instance on the sense strand. Theresulting RNAi agents present superior gene silencing activity.

The inventors surprisingly discovered that when the sense strand andantisense strand of the RNAi agent are completely modified, having oneor more motifs of three identical modifications on three consecutivenucleotides at or near the cleavage site of at least one strand of aRNAi agent superiorly enhanced the gene silencing activity of the RNAiagent.

Accordingly, the invention provides RNAi agents, e.g., double strandedRNAi agents, capable of inhibiting the expression of a target gene(i.e., a TTR gene) in vivo. The RNAi agent comprises a sense strand andan antisense strand. Each strand of the RNAi agent can range from 12-30nucleotides in length. For example, each strand can be between 14-30nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides inlength, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides inlength, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups of RNAi agent at 3′-end, or 5′-end or bothends of a strand. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be other sequence. The first and second strands can also be joined,e.g., by additional bases to form a hairpin, or by other non-baselinkers.

The RNAi agents provided by the present invention include agents withchemical modifications as disclosed, for example, in U.S. ProvisionalApplication No. 61/561,710, filed on Nov. 18, 2011, InternationalApplication No. PCT/US2011/051597, filed on Sep. 15, 2010, and PCTPublication WO 2009/073809, the entire contents of each of which areincorporated herein by reference.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be other sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region contains two nucleotides having a phosphorothioatebetween the two nucleotides, where the two nucleotides can be the sameor different. In one embodiment, the overhang is present at the 3′-endof the sense strand, antisense strand or both strands. In oneembodiment, this 3′-overhang is present in the antisense strand. In oneembodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang is located at the3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilethe Applicants are not bound by theory, the theoretical mechanism isthat the asymmetric blunt end at the 5′-end of the antisense strand and3′-end overhang of the antisense strand favor the guide strand loadinginto RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19 nt inlength, wherein the sense strand contains at least one motif of three2′-F modifications on three consecutive nucleotides at positions 7,8,9from the 5′end. The antisense strand contains at least one motif ofthree 2′-O-methyl modifications on three consecutive nucleotides atpositions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent is a double ended bluntmer of 20 nt inlength, wherein the sense strand contains at least one motif of three2′-F modifications on three consecutive nucleotides at positions 8,9,10from the 5′end. The antisense strand contains at least one motif ofthree 2′-O-methyl modifications on three consecutive nucleotides atpositions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent is a double ended bluntmer of 21 nt inlength, wherein the sense strand contains at least one motif of three2′-F modifications on three consecutive nucleotides at positions 9, 10,11 from the 5′end. The antisense strand contains at least one motif ofthree 2′-O-methyl modifications on three consecutive nucleotides atpositions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotides (nt) sensestrand and a 23 nucleotides (nt) antisense strand, wherein the sensestrand contains at least one motif of three 2′-F modifications on threeconsecutive nucleotides at positions 9,10,11 from the 5′end; theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at positions 11, 12, 13from the 5′end, wherein one end of the RNAi agent is blunt, while theother end comprises a 2 nt overhang. Preferably, the 2 nt overhang is atthe 3′-end of the antisense. Optionally, the RNAi agent furthercomprises a ligand (preferably GalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5′ terminal nucleotide (position 1) positions1 to 23 of the first strand comprise at least 8 ribonucleotides;antisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3′terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3′ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell; and whereinthe sense strand contains at least one motif of three 2′-F modificationson three consecutive nucleotides, where at least one of the motifsoccurs at or near the cleavage site. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11,12,13 from the 5′ end; wherein the 3′ end of the first strand and the5′ end of the second strand form a blunt end and the second strand is1-4 nucleotides longer at its 3′ end than the first strand, wherein theduplex region which is at least 25 nucleotides in length, and the secondstrand is sufficiently complementary to a target mRNA along at least 19nt of the second strand length to reduce target gene expression when theRNAi agent is introduced into a mammalian cell, and wherein dicercleavage of the RNAi agent preferentially results in an siRNA comprisingthe 3′ end of the second strand, thereby reducing expression of thetarget gene in the mammal. Optionally, the RNAi agent further comprisesa ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For RNAi agent having a duplex region of 17-23 nt in length, thecleavage site of the antisense strand is typically around the 10, 11 and12 positions from the 5′-end. Thus, the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1′nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1′ paired nucleotide within the duplex region from the5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif should occur at or near the cleavage siteof the strand and the other motifs may be wing modifications. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thanthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain at least two motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that is present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or2′-fluoro. The strands can contain more than one modification. In oneembodiment, each residue of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisense strand may start with “BBAABBAA” from5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both in anyposition of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand or antisense strand; or thesense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand.

In one embodiment, the RNAi comprises the phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within duplex region. For example, at least 2, 3, 4, or allthe overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. Preferably, these terminal three nucleotides may be at the3′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch canoccur in the overhang region or the duplex region. The base pair can beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand can be chosen independently from the group of: A:U,G:U, I:C, and mismatched pairs, e.g., non-canonical or other thancanonical pairings or pairings which include a universal base, topromote the dissociation of the antisense strand at the 5′-end of theduplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair. In one embodiment, the sense strand sequence may berepresented by formula (I):

5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and XXX, YYY andZZZ each independently represent one motif of three identicalmodifications on three consecutive nucleotides. Preferably YYY is all2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of-the sensestrand, the count starting from the 1′ nucleotide, from the 5′-end; oroptionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Ia);

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′  (Ib); or

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Ic).

When the sense strand is represented by formula (Ia), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′n _(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23 ntin length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11,12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, withthe count starting from the 1′ nucleotide, from the 5′-end; oroptionally, the count starting at the 1′ paired nucleotide within theduplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs atpositions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and 1 is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n _(p′)3′  (IIa);

5′n _(q′)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n _(p′)3′  (IIb); or

5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p′)3′  (IIc).

When the antisense strand is represented by formula (IIa), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, 2′-deoxy or 2′-fluoro.For example, each nucleotide of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′,Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1′ nucleotide from the5′-end, or optionally, the count starting at the 1 paired nucleotidewithin the duplex region, from the 5′-end; and Y represents 2′-Fmodification. The sense strand may additionally contain XXX motif or ZZZmotifs as wing modifications at the opposite end of the duplex region;and XXX and ZZZ each independently represents a 2′-OMe modification or2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1 nucleotide from the 5′-end, or optionally, the count starting atthe 1′ paired nucleotide within the duplex region, from the 5′-end; andY′ represents 2′-O-methyl modification. The antisense strand mayadditionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modificationsat the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ eachindependently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib)and (Ic) forms a duplex with a antisense strand being represented by anyone of formulas (Ha), (IIb) and (IIc), respectively.

Accordingly, the RNAi agents of the invention may comprise a sensestrand and an antisense strand, each strand having 14 to 30 nucleotides,the RNAi duplex represented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′   (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q) independently represents anoverhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both iand j are 1. In another embodiment, k is 1 and l is 0; k is 0 and 1 is1; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a) n _(q)′5′   (IIIa)

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n _(q)′5′   (IIIb)

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n _(q)′5′  (IIc)

When the RNAi agent is represented by formula (IIIa), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIb), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and (IIIc) may bethe same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb) or(IIIc), at least one of the Y nucleotides may form a base pair with oneof the Y′ nucleotides. Alternatively, at least two of the Y nucleotidesform base pairs with the corresponding Y′ nucleotides; or all three ofthe Y nucleotides all form base pairs with the corresponding Y′nucleotides.

When the RNAi agent is represented by formula (IIIa) or (IIIc), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIb) or (IIIc), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), whereinthe duplexes are connected by a linker. The linker can be cleavable ornon-cleavable. Optionally, the multimer further comprise a ligand. Eachof the duplexes can target the same gene or two different genes; or eachof the duplexes can target same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb)or (IIIc), wherein the duplexes are connected by a linker. The linkercan be cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb) or (IIIc) are linked to each other at the 5′ end, and one or bothof the 3′ ends of the are optionally conjugated to a ligand. Each of theagents can target the same gene or two different genes; or each of theagents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents. Such publicationsinclude WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511,WO2007/117686, WO2009/014887 and WO2011/031520 the entire contents ofwhich are hereby incorporated herein by reference.

The RNAi agent that contains conjugations of one or more carbohydratemoieties to a RNAi agent can optimize one or more properties of the RNAiagent. In many cases, the carbohydrate moiety will be attached to amodified subunit of the RNAi agent. For example, the ribose sugar of oneor more ribonucleotide subunits of a dsRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier towhich is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent of the invention is anagent selected from the group of agents listed in Table 1 and consistingof D1000, D1001, D1002, D1003, D1004, D1005, D1006, D1007, D1008, D1009,D1010, D1011, D1012, D1013, D1014, D1015, D1016, D1017, D1018, D1019,D1020, D1021, D1022, D1023, D1024, D1025, D1026, D1027, D1028, D1029,D1030, D1031, D1032, D1033, D1034, D1035, D1036, D1037, D1038, D1039,D1040, D1041, D1042, D1043, D1044, D1045, D1046, D1047, D1048, D1049,D1050, D1051, D1052, D1053, D1054, D1055, D1056, D1057, D1058, D1059,D1060, D1061, D1062, D1063, D1064, D1065, D1066, D1067, D1068, D1069,D1070, D1071, D1072, D1073, D1074, D1075, D1076, D1077, D1078, D1079,D1080, D1081, D1082, D1083, D1084, D1085, D1086, D1087, D1088, D1089,D1090, D1091, D1092, D1093, D1094, D1095, D1096, D1097, D1098, D1099,D1100, D1101, D1102, D1103, D1104, D1105, D1106, D1107, D1108, D1109,D1110, D1111, D1112, D1113, D1114, D1115, D1116, D1117, D1118, D1119,D1120, D1121, D1122, D1123, D1124, D1125, D1126, D1127, D1128, D1129,D1130, D1131, D1132, D1133, D1134, D1135, D1136, D1137, D1138, D1139,D1140, D1141, D1142, D1143, D1144, D1145, D1146, D1147, D1148, D1149,D1150, D1151, D1152, D1153, D1154, D1155, D1156, D1157, D1158, D1159,D1160, D1161, D1162, D1163, D1164, D1165, D1166, D1167, D1168, D1169,D1170, D1171, D1172, D1173, D1174, D1175, D1176, D1177, D1178, D1179,D1180, D1181, D1182, D1183, D1184, D1185, D1186, D1187, D1188, D1189,D1190, D1191, D1192, D1193, D1194, D1195, D1196, D1197, D1198, D1199,D1200, D1201, D1202, D1203, D1204, D1205, D1206, D1207, D1208, D1209,D1210, D1211, D1212, D1213, D1214, D1215, D1216, D1217, D1218, D1219,D1220, D1221, D1222, D1223, D1224, D1225, D1226, D1227, D1228, D1229,D1230, D1231, D1232, D1233, D1234, D1235, D1236, D1237, D1238, D1239,D1240, D1241, D1242, D1243, D1244, D1245, D1246, D1247, D1248, D1249,D1250, D1251, D1252, D1253, D1254, D1255, D1256, D1257, D1258, D1259,D1260, D1261, D1262, D1263, D1264, D1265, D1266, D1267, D1268, D1269,D1270, D1271, D1272, D1273, D1274, D1275, D1276, D1277, D1278, D1279,D1280, D1281, D1282, D1283, D1284, D1285, D1286, D1287, D1288, D1289,D1290, D1291, D1292, D1293, D1294, D1295, D1296, D1297, D1298, D1299,D1300, D1301, D1302, D1303, D1304, D1305, D1306, D1307, D1308, D1309,D1310, D1311, D1312, D1313, D1314, D1315, D1316, D1317, D1318, D1319,D1320, D1321, D1322, D1323, D1324, D1325, D1326, D1327, D1328, D1329,D1330, D1331, D1332, D1333, D1334, D1335, D1336, D1337, D1338, D1339,D1340, D1341, D1342, D1343, D1344, D1345, D1346, D1347, D1348, D1349,D1350, D1351, D1352, D1353, D1354, D1355, D1356, D1357, D1358, D1359,D1360, D1361, D1362, D1363, D1364, D1365, D1366, D1367, D1368, D1369,D1370, D1371, D1372, D1373, D1374, D1375, D1376, D1377, D1378, D1379,D1380, D1381, D1382, D1383, D1384, D1385, D1386, D1387, D1388, D1389,D1390, D1391, D1392, D1393, D1394, D1395, D1396, D1397, D1398, D1399,D1400, D1401, D1402, D1403, D1404, D1405, D1406, D1407, D1408, D1409,D1410, D1411, D1412, D1413, D1414, D1415, D1416, D1417, D1418, D1419,D1420, D1421, D1422, D1423, D1424, D1425, D1426, D1427, D1428, D1429,D1430, D1431, D1432, D1433, D1434, D1435, D1436, D1437, D1438, D1439,D1440, D1441, D1442, D1443, D1444, D1445, D1446, D1447, D1448, D1449,D1450, D1451, D1452, D1453, D1454, D1455, D1456, D1457, D1458, D1459,D1460, D1461, D1462, D1463, D1464, D1465, D1466, D1467, D1468, D1469,D1470, D1471, D1472, D1473, D1474, D1475, D1476, D1477, D1478, D1479,D1480, D1481, D1482, D1483, D1484, D1485, D1486, D1487, D1488, D1489,D1490, D1491, D1492, D1493, D1494, D1495, D1496, D1497, D1498, D1499,D1500, D1501, D1502, D1503, D1504, D1505, D1506, D1507, D1508, D1509,D1510, D1511, D1512, D1513, D1514, D1515, D1516, D1517, D1518, D1519,D1520, D1521, D1522, D1523, D1524, D1525, D1526, D1527, D1528, D1529,D1530, D1531, D1532, D1533, D1534, D1535, D1536, D1537, D1538, D1539,D1540, D1541, D1542, D1543, D1544, D1545, D1546, D1547, D1548, D1549,D1550, D1551, D1552, D1553, D1554, D1555, D1556, D1557, D1558, D1559,D1560, D1561, D1562, D1563, D1564, D1565, D1566, D1567, D1568, D1569,D1570, D1571, D1572, D1573, D1574, D1575, D1576, D1577, D1578, D1579,D1580, D1581, D1582, D1583, D1584, D1585, D1586, D1587, D1588, D1589,D1590, D1591, D1592, D1593, D1594, D1595, D1596, D1597, D1598, D1599,D1600, D1601, D1602, D1603, D1604, D1605, D1606, D1607, D1608, D1609,D1610, D1611, D1612, D1613, D1614, D1615, D1616, D1617, D1618, D1619,D1620, D1621, D1622, D1623, D1624, D1625, D1626, D1627, D1628, D1629,D1630, D1631, D1632, D1633, D1634, D1635, D1636, D1637, D1638, D1639,D1640, D1641, D1642, D1643, D1644, D1645, D1646, D1647, D1648, D1649,D1650, D1651, D1652, D1653, D1654, D1655, D1656, D1657, D1658, D1659,D1660, D1661, D1662, D1663, D1664, D1665, D1666, D1667, D1668, D1669,D1670, D1671, D1672, D1673, D1674, D1675, D1676, D1677, D1678, D1679,D1680, D1681, D1682, D1683, D1684, D1685, D1686, D1687, D1688, D1689,D1690, D1691, D1692, D1693, D1694, D1695, D1696, D1697, D1698, D1699,D1700, D1701, D1702, D1703, D1704, D1705, D1706, D1707, D1708, D1709,D1710, D1711, D1712, D1713, D1714, D1715, D1716, D1717, D1718, D1719,D1720, D1721, D1722, D1723, D1724, D1725, D1726, D1727, D1728, D1729,D1730, D1731, D1732, D1733, D1734, D1735, D1736, D1737, D1738, D1739,D1740, D1741, D1742, D1743, D1744, D1745, D1746, D1747, D1748, D1749,D1750, D1751, D1752, D1753, D1754, D1755, D1756, D1757, D1758, D1759,D1760, D1761, D1762, D1763, D1764, D1765, D1766, D1767, D1768, D1769,D1770, D1771, D1772, D1773, D1774, D1775, D1776, D1777, D1778, D1779,D1780, D1781, D1782, D1783, D1784, D1785, D1786, D1787, D1788, D1789,D1790, D1791, D1792, D1793, D1794, D1795, D1796, D1797, D1798, D1799,D1800, D1801, D1802, D1803, D1804, D1805, D1806, D1807, D1808, D1809,D1810, D1811, D1812, D1813, D1814, D1815, D1816, D1817, D1818, D1819,D1820, D1821, D1822, D1823, D1824, D1825, D1826, D1827, D1828, D1829,D1830, D1831, D1832, D1833, D1834, D1835, D1836, D1837, D1838, D1839,D1840, D1841, D1842, D1843, D1844, D1845, D1846, D1847, D1848, D1849,D1850, D1851, D1852, D1853, D1854, D1855, D1856, D1857, D1858, D1859,D1860, D1861, D1862, D1863, D1864, D1865, D1866, D1867, D1868, D1869,D1870, D1871, D1872, D1873, D1874, D1875, D1876, D1877, D1878, D1879,D1880, D1881, D1882, D1883, D1884, D1885, D1886, D1887, D1888, D1889,D1890, D1891, D1892, D1893, D1894, D1895, D1896, D1897, D1898, D1899,D1900, D1901, D1902, D1903, D1904, D1905, D1906, D1907, D1908, D1909,D1910, D1911, D1912, D1913, D1914, D1915, D1916, D1917, D1918, D1919,D1920, D1921, D1922, D1923, D1924, D1925, D1926, D1927, D1928, D1929,D1930, D1931, D1932, D1933, D1934, D1935, D1936, D1937, D1938, D1939,D1940, D1941, D1942, D1943, D1944, D1945, D1946, D1947, D1948, D1949,D1950, D1951, D1952, D1953, D1954, D1955, D1956, D1957, D1958, D1959,D1960, D1961, D1962, D1963, D1964, D1965, D1966, D1967, D1968, D1969,D1970, D1971, D1972, D1973, D1974, D1975, D1976, D1977, D1978, D1979,D1980, D1981, D1982, D1983, D1984, D1985, D1986, D1987, D1988, D1989,D1990, D1991, D1992, D1993, D1994, D1995, D1996, D1997, D1998, D1999,D2000, D2001, D2002, D2003, D2004, D2005, D2006, D2007, D2008, D2009,D2010, D2011, D2012, D2013, D2014, D2015, D2016, D2017, D2018, D2019,D2020, D2021, D2022, D2023, D2024, D2025, D2026, D2027, D2028, D2029,D2030, D2031, D2032, D2033, D2034, D2035, D2036, D2037, D2038, D2039,D2040, D2041, D2042, D2043, D2044, D2045, D2046, D2047, D2048, D2049,D2050, D2051, D2052, D2053, D2054, D2055, D2056, D2057, D2058, D2059,D2060, D2061, D2062, D2063, D2064, D2065, D2066, D2067, D2068, D2069,D2070, D2071, D2072, D2073, D2074, D2075, D2076, D2077, D2078, D2079,D2080, D2081, D2082, D2083, D2084, D2085, D2086, D2087, D2088, D2089,D2090 and D2091.

These agents may further comprise a ligand, such as a GalNAc ligand.

Ligands

The RNAi agents of the invention, e.g., double stranded RNAi agents, mayoptionally be conjugated to one or more ligands. The ligand can beattached to the sense strand, antisense strand or both strands, at the3′-end, 5′-end or both ends. For instance, the ligand may be conjugatedto the sense strand. In preferred embodiments, the ligand is conjugatedto the 3′-end of the sense strand. In one preferred embodiment, theligand is a GalNAc ligand. In particularly preferred embodiments, theligand is GalNAc₃:

A wide variety of entities can be coupled to the RNAi agents of thepresent invention. Preferred moieties are ligands, which are coupled,preferably covalently, either directly or indirectly via an interveningtether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, receptor e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand. Ligands providingenhanced affinity for a selected target are also termed targetingligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In one embodiment, the endosomolytic ligand assumes itsactive conformation at endosomal pH. The “active” conformation is thatconformation in which the endosomolytic ligand promotes lysis of theendosome and/or transport of the composition of the invention, or itscomponents, from the endosome to the cytoplasm of the cell. Exemplaryendosomolytic ligands include the GALA peptide (Subbarao et al.,Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J.Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, theendosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched.

Ligands can improve transport, hybridization, and specificity propertiesand may also improve nuclease resistance of the resultant natural ormodified oligoribonucleotide, or a polymeric molecule comprising anycombination of monomers described herein and/or natural or modifiedribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g., an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Other examples of ligands include dyes, intercalating agents (e.g.,acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases or a chelator(e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholanic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]z, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.,biotin), transport/absorption facilitators (e.g., aspirin, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the oligonucleotide into the cellby, for example, activating an inflammatory response. Exemplary ligandsthat would have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, naproxen or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. In another alternative, the peptide moiety caninclude a hydrophobic membrane translocation sequence (MTS). Anexemplary hydrophobic MTS-containing peptide is RFGF having the aminoacid sequence AAVALLPAVLLALLAP (SEQ ID NO:4). An RFGF analogue (e.g.,amino acid sequence AALLPVLLAAP) (SEQ ID NO:5) containing a hydrophobicMTS can also be a targeting moiety. The peptide moiety can be a“delivery” peptide, which can carry large polar molecules includingpeptides, oligonucleotides, and protein across cell membranes. Forexample, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ IDNO:6) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ IDNO:7) have been found to be capable of functioning as delivery peptides.A peptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated monomer unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized. An RGD peptide moiety can be used to target a tumor cell, suchas an endothelial tumor cell or a breast cancer tumor cell (Zitzmann etal., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitatetargeting of an iRNA agent to tumors of a variety of other tissues,including the lung, kidney, spleen, or liver (Aoki et al., Cancer GeneTherapy 8:783-787, 2001). Preferably, the RGD peptide will facilitatetargeting of an iRNA agent to the kidney. The RGD peptide can be linearor cyclic, and can be modified, e.g., glycosylated or methylated tofacilitate targeting to specific tissues. For example, a glycosylatedRGD peptide can deliver an iRNA agent to a tumor cell expressing αvß₃(Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides thattarget markers enriched in proliferating cells can be used. For example,RGD containing peptides and peptidomimetics can target cancer cells, inparticular cells that exhibit an integrin. Thus, one could use RGDpeptides, cyclic peptides containing RGD, RGD peptides that includeD-amino acids, as well as synthetic RGD mimics. In addition to RGD, onecan use other moieties that target the integrin ligand. Generally, suchligands can be used to control proliferating cells and angiogeneis.Preferred conjugates of this type of ligand target PECAM-1, VEGF, orother cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide can be an amphipathic α-helicalpeptide. Exemplary amphipathic α-helical peptides include, but are notlimited to, cecropins, lycotoxins, paradaxins, buforin, CPF,bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clavapeptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, HzApeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an aptamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulators includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Exemplary PK modulatorsinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.,oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple phosphorothioate linkages in the backbone are alsoamenable to the present invention as ligands (e.g., as PK modulatingligands).

In addition, aptamers that bind serum components (e.g., serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S.patent applications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S.Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934,filed Aug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 andU.S. Ser. No. 11/944,227 filed Nov. 21, 2007, which are incorporated byreference in their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether, e.g., a carrier described herein. The ligand ortethered ligand may be present on a monomer when the monomer isincorporated into the growing strand. In some embodiments, the ligandmay be incorporated via coupling to a “precursor” monomer after the“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporatedinto a growing oligonucleotide strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor monomer bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction may be incorporated, e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used,although the ligand is typically a carbohydrate e.g. monosaccharide(such as GalNAc), disaccharide, trisaccharide, tetrasaccharide,polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include thosediscussed above. For example, the ligand can be one or more GalNAc(N-acetylglucosamine) derivatives attached through a bivalent ortrivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to abivalent and trivalent branched linkers include the structures shown inany of formula (IV)-(VII):

wherein:

q^(2A), q^(2B), q^(3A), q^(3B), q^(4A), q^(4B), q^(5A), q^(5B) andq^(5C) represent independently for each occurrence 0-20 and wherein therepeating unit can be the same or different; p^(2A), p^(2B), p^(3A),p^(3B), p^(4A), p^(4B), p^(5A), p^(5B), p^(5C), T^(2A), T^(2B), T^(3A),T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independentlyfor each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH orCH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and

R^(a) is H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for usewith RNAi agents for inhibiting the expression of a target gene, such asthose of formula (VII):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative. Examples of suitable bivalent and trivalent branchedlinker groups conjugating GalNAc derivatives include but are not limitedto the following compounds:

In other embodiments, the RNAi agent of the invention is an agentselected from the group consisting of AD-45163, AD-45165, AD-51544,AD-51545, AD-51546, and AD-51547.

III. Pharmaceutical Compositions

The RNAi agents of the invention may be formulated for administration inany convenient way for use in human or veterinary medicine, by analogywith other pharmaceuticals. The pharmaceutical compositions comprisingRNAi agents of the invention may be, for example, solutions with orwithout a buffer, or compositions containing pharmaceutically acceptablecarriers. Such compositions include, for example, aqueous or crystallinecompositions, liposomal formulations, micellar formulations, emulsions,and gene therapy vectors.

In the methods of the invention, the RNAi agent may be administered in asolution. A free RNAi agent may be administered in an unbufferedsolution, e.g., in saline or in water. Alternatively, the free siRNA mayalso be administered in a suitable buffer solution. The buffer solutionmay comprise acetate, citrate, prolamine, carbonate, or phosphate, orany combination thereof. In a preferred embodiment, the buffer solutionis phosphate buffered saline (PBS). The pH and osmolarity of the buffersolution containing the RNAi agent can be adjusted such that it issuitable for administering to a subject.

In some embodiments, the buffer solution further comprises an agent forcontrolling the osmolarity of the solution, such that the osmolarity iskept at a desired value, e.g., at the physiologic values of the humanplasma. Solutes which can be added to the buffer solution to control theosmolarity include, but are not limited to, proteins, peptides, aminoacids, non-metabolized polymers, vitamins, ions, sugars, metabolites,organic acids, lipids, or salts. In some embodiments, the agent forcontrolling the osmolarity of the solution is a salt. In certainembodiments, the agent for controlling the osmolarity of the solution issodium chloride or potassium chloride.

In other embodiments, the RNAi agent is formulated as a composition thatincludes one or more RNAi agents and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

In one embodiment, the RNAi agent preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA). Forexample, an RNAi agent composition for the treatment of a TTR-associateddisease, e.g., a transthyretin-related hereditary amyloidosis (familialamyloid polyneuropathy, FAP), may include a known drug for theamelioration of FAP, e.g., Tafamidis (INN, or Fx-1006A or Vyndaqel).

A formulated RNAi agent composition can assume a variety of states. Insome examples, the composition is at least partially crystalline,uniformly crystalline, and/or anhydrous (e.g., it contains less than 80,50, 30, 20, or 10% of water). In another example, the RNAi agent is inan aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can be incorporatedinto a delivery vehicle, e.g., a liposome (particularly for the aqueousphase) or a particle (e.g., a microparticle as can be appropriate for acrystalline composition). Generally, the RNAi agent composition isformulated in a manner that is compatible with the intended method ofadministration, as described herein. For example, in particularembodiments the composition is prepared by at least one of the followingmethods: spray drying, lyophilization, vacuum drying, evaporation, fluidbed drying, or a combination of these techniques; or sonication with alipid, freeze-drying, condensation and other self-assembly.

An RNAi agent preparation can be formulated in combination with anotheragent, e.g., another therapeutic agent or an agent that stabilizes RNAiagent, e.g., a protein that complexes with the RNAi agent to form aniRNP. Still other agents include chelators, e.g., EDTA (e.g., to removedivalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broadspecificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the RNAi agent preparation includes another siRNAcompound, e.g., a second RNAi agent that can mediate RNAi with respectto a second gene, or with respect to the same gene. Still otherpreparation can include at least 3, 5, ten, twenty, fifty, or a hundredor more different RNAi agent species. Such RNAi agents can mediate RNAiwith respect to a similar number of different genes.

The iRNA agents of the invention may be formulated for pharmaceuticaluse. Pharmaceutically acceptable compositions comprise atherapeutically- or prophylactically effective amount of one or more ofthe dsRNA agents in any of the preceding embodiments, taken alone orformulated together with one or more pharmaceutically acceptablecarriers (additives), excipient and/or diluents.

Methods of preparing pharmaceutical compositions of the inventioninclude the step of bringing into association an RNAi agent of thepresent invention with the carrier and, optionally, one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing into association an RNAi agent of thepresent invention with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

The pharmaceutical compositions may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally. Delivery using subcutaneous or intravenous methods can beparticularly advantageous.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the compositionand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium state, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of RNAi agent which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, and the particular mode of administration. The RNAi agent whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the RNAi agent which produces a desiredeffect, e.g., therapeutic or prophylactic effect. Generally, out of onehundred percent, this amount will range from about 0.1 percent to aboutninety-nine percent of RNAi agent, preferably from about 5 percent toabout 70 percent, most preferably from about 10 percent to about 30percent.

In certain embodiments, a composition of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and an RNAiagent of the present invention. In certain embodiments, anaforementioned composition renders orally bioavailable an RNAi agent ofthe present invention.

In some cases, in order to prolong the effect of an RNAi agent, it isdesirable to slow the absorption of the agent from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the RNAi agent then depends uponits rate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered RNAi agent may be accomplished by dissolvingor suspending the agent in an oil vehicle.

Liposomes

An RNAi agent of the invention can be formulated for delivery in amembranous molecular assembly, e.g., a liposome or a micelle. As usedherein, the term “liposome” refers to a vesicle composed of amphiphiliclipids arranged in at least one bilayer, e.g., one bilayer or aplurality of bilayers. Liposomes include unilamellar and multilamellarvesicles that have a membrane formed from a lipophilic material and anaqueous interior. The aqueous portion contains the RNAi agentcomposition. The lipophilic material isolates the aqueous interior froman aqueous exterior, which typically does not include the RNAi agentcomposition, although in some examples, it may. Liposomes are useful forthe transfer and delivery of active ingredients to the site of action.Because the liposomal membrane is structurally similar to biologicalmembranes, when liposomes are applied to a tissue, the liposomal bilayerfuses with bilayer of the cellular membranes. As the merging of theliposome and cell progresses, the internal aqueous contents that includethe RNAi agent are delivered into the cell where the RNAi agent canspecifically bind to a target RNA and can mediate RNAi. In some casesthe liposomes are also specifically targeted, e.g., to direct the RNAiagent to particular cell types.

A liposome containing an RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also be adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes that are pH-sensitive or negatively-charged entrap nucleicacid molecules rather than complex with them. Since both the nucleicacid molecules and the lipid are similarly charged, repulsion ratherthan complex formation occurs. Nevertheless, some nucleic acid moleculesare entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include RNAi agent can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transfersomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transfersomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Surfactants

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes (see above). RNAi agent (or aprecursor, e.g., a larger dsiRNA which can be processed into a siRNA, ora DNA which encodes a siRNA or precursor) compositions can include asurfactant. In one embodiment, the siRNA is formulated as an emulsionthat includes a surfactant. The most common way of classifying andranking the properties of the many different types of surfactants, bothnatural and synthetic, is by the use of the hydrophile/lipophile balance(HLB). The nature of the hydrophilic group provides the most usefulmeans for categorizing the different surfactants used in formulations(Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., NewYork, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical products and are usable over a wide range of pH values.In general their HLB values range from 2 to about 18 depending on theirstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters.Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and Other Membranous Formulations

The RNAi agents of the invention can also be provided as micellarformulations. “Micelles” are defined herein as a particular type ofmolecular assembly in which amphipathic molecules are arranged in aspherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compound. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

Particles

In another embodiment, an RNAi agent of the invention may beincorporated into a particle, e.g., a microparticle. Microparticles canbe produced by spray-drying, but may also be produced by other methodsincluding lyophilization, evaporation, fluid bed drying, vacuum drying,or a combination of these techniques.

IV. Methods for Inhibiting TTR Expression

The present invention also provides methods of inhibiting expression ofa transthyretin (TTR) in a cell. The methods include contacting a cellwith an RNAi agent, e.g., double stranded RNAi agent, in an amounteffective to inhibit expression of TTR in the cell, thereby inhibitingexpression of TTR in the cell.

Contacting of a cell with an RNAi agent, e.g., a double stranded RNAiagent, may be done in vitro or in vivo. Contacting a cell in vivo withthe RNAi agent includes contacting a cell or group of cells within asubject, e.g., a human subject, with the RNAi agent. Combinations of invitro and in vivo methods of contacting a cell are also possible.Contacting a cell may be direct or indirect, as discussed above.Furthermore, contacting a cell may be accomplished via a targetingligand, including any ligand described herein or known in the art. Inpreferred embodiments, the targeting ligand is a carbohydrate moiety,e.g., a GalNAc₃ ligand, or any other ligand that directs the RNAi agentto a site of interest, e.g., the liver of a subject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating”, “suppressing”, and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a TTR” is intended to refer toinhibition of expression of any TTR gene (such as, e.g., a mouse TTRgene, a rat TTR gene, a monkey TTR gene, or a human TTR gene) as well asvariants or mutants of a TTR gene. Thus, the TTR gene may be a wild-typeTTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise toamyloid deposition), or a transgenic TTR gene in the context of agenetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a TTR gene” includes any level of inhibitionof a TTR gene, e.g., at least partial suppression of the expression of aTTR gene. The expression of the TTR gene may be assessed based on thelevel, or the change in the level, of any variable associated with TTRgene expression, e.g., TTR mRNA level, TTR protein level, or the numberor extent of amyloid deposits. This level may be assessed in anindividual cell or in a group of cells, including, for example, a samplederived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with TTR expressioncompared with a control level. The control level may be any type ofcontrol level that is utilized in the art, e.g., a pre-dose baselinelevel, or a level determined from a similar subject, cell, or samplethat is untreated or treated with a control (such as, e.g., buffer onlycontrol or inactive agent control).

In some embodiments of the methods of the invention, expression of a TTRgene is inhibited by at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%. at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%.

Inhibition of the expression of a TTR gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which a TTR gene is transcribed and which has or have beentreated (e.g., by contacting the cell or cells with an RNAi agent of theinvention, or by administering an RNAi agent of the invention to asubject in which the cells are or were present) such that the expressionof a TTR gene is inhibited, as compared to a second cell or group ofcells substantially identical to the first cell or group of cells butwhich has not or have not been so treated (control cell(s)). Inpreferred embodiments, the inhibition is assessed by expressing thelevel of mRNA in treated cells as a percentage of the level of mRNA incontrol cells, using the following formula:

$\frac{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} ) - ( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} )}{( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} )}{\bullet 100}\%$

Alternatively, inhibition of the expression of a TTR gene may beassessed in terms of a reduction of a parameter that is functionallylinked to TTR gene expression, e.g., TTR protein expression, retinolbinding protein level, vitamin A level, or presence of amyloid depositscomprising TTR. TTR gene silencing may be determined in any cellexpressing TTR, either constitutively or by genomic engineering, and byany assay known in the art. The liver is the major site of TTRexpression. Other significant sites of expression include the choroidplexus, retina and pancreas.

Inhibition of the expression of a TTR protein may be manifested by areduction in the level of the TTR protein that is expressed by a cell orgroup of cells (e.g., the level of protein expressed in a sample derivedfrom a subject). As explained above for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of a TTR gene includes a cell or group ofcells that has not yet been contacted with an RNAi agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of TTR mRNA that is expressed by a cell or group of cells, orthe level of circulating TTR mRNA, may be determined using any methodknown in the art for assessing mRNA expression. In one embodiment, thelevel of expression of TTR in a sample is determined by detecting atranscribed polynucleotide, or portion thereof, e.g., mRNA of the TTRgene. RNA may be extracted from cells using RNA extraction techniquesincluding, for example, using acid phenol/guanidine isothiocyanateextraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen)or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizingribonucleic acid hybridization include nuclear run-on assays, RT-PCR,RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035),Northern blotting, in situ hybridization, and microarray analysis.Circulating TTR mRNA may be detected using methods the described inPCT/JS2012/043584, the entire contents of which are hereby incorporatedherein by reference.

In one embodiment, the level of expression of TTR is determined using anucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific TTR.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to TTRmRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of TTR mRNA.

An alternative method for determining the level of expression of TTR ina sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of TTR isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System).

The expression levels of TTR mRNA may be monitored using a membrane blot(such as used in hybridization analysis such as Northern, Southern, dot,and the like), or microwells, sample tubes, gels, beads or fibers (orany solid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of TTR expressionlevel may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of TTR protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, Western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention can bemonitored by detecting or monitoring a reduction in an amyloid TTRdeposit. Reducing an amyloid TTR deposit, as used herein, includes anydecrease in the size, number, or severity of TTR deposits, or to aprevention or reduction in the formation of TTR deposits, within anorgan or area of a subject, as may be assessed in vitro or in vivo usingany method known in the art. For example, some methods of assessingamyloid deposits are described in Gertz, M. A. & Rajukumar, S. V.(Editors) (2010), Amyloidosis: Diagnosis and Treatment, New York: HumanaPress. Methods of assessing amyloid deposits may include biochemicalanalyses, as well as visual or computerized assessment of amyloiddeposits, as made visible, e.g., using immunohistochemical staining,fluorescent labeling, light microscopy, electron microscopy,fluorescence microscopy, or other types of microscopy. Invasive ornoninvasive imaging modalities, including, e.g., CT, PET, or NMR/MRIimaging may be employed to assess amyloid deposits.

The methods of the invention may reduce TTR deposits in any number oftissues or regions of the body including but not limited to the heart,liver, spleen, esophagus, stomach, intestine (ileum, duodenum andcolon), brain, sciatic nerve, dorsal root ganglion, kidney and retina.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes), the retina or parts of the retina (e.g., retinal pigmentepithelium), the central nervous system or parts of the central nervoussystem (e.g., ventricles or choroid plexus), or the pancreas or certaincells or parts of the pancreas. In preferred embodiments, a “samplederived from a subject” refers to blood or plasma drawn from thesubject. In further embodiments, a “sample derived from a subject”refers to liver tissue or retinal tissue derived from the subject.

In some embodiments of the methods of the invention, the RNAi agent isadministered to a subject such that the RNAi agent is delivered to aspecific site within the subject. The inhibition of expression of TTRmay be assessed using measurements of the level or change in the levelof TTR mRNA or TTR protein in a sample derived from fluid or tissue fromthe specific site within the subject. In preferred embodiments, the siteis selected from the group consisting of liver, choroid plexus, retina,and pancreas. The site may also be a subsection or subgroup of cellsfrom any one of the aforementioned sites (e.g., hepatocytes or retinalpigment epithelium). The site may also include cells that express aparticular type of receptor (e.g., hepatocytes that express theasialoglycoprotein receptor).

V. Methods for Treating or Preventing a TTR-Associated Disease

The present invention also provides methods for treating or preventing aTTR-associated disease in a subject. The methods include administeringto the subject a therapeutically effective amount or prophylacticallyeffective amount of an RNAi agent of the invention.

As used herein, a “subject” includes either a human or a non-humananimal, preferably a vertebrate, and more preferably a mammal. A subjectmay include a transgenic organism. Most preferably, the subject is ahuman, such as a human suffering from or predisposed to developing aTTR-associated disease.

In some embodiments, the subject is suffering from a TTR-associateddisease. In other embodiments, the subject is a subject at risk fordeveloping a TTR-associated disease, e.g., a subject with a TTR genemutation that is associated with the development of a TTR associateddisease, a subject with a family history of TTR-associated disease, or asubject who has signs or symptoms suggesting the development of TTRamyloidosis.

A “TTR-associated disease,” as used herein, includes any disease causedby or associated with the formation of amyloid deposits in which thefibril precursors consist of variant or wild-type TTR protein. Mutantand wild-type TTR give rise to various forms of amyloid deposition(amyloidosis). Amyloidosis involves the formation and aggregation ofmisfolded proteins, resulting in extracellular deposits that impairorgan function. Clinical syndromes associated with TTR aggregationinclude, for example, senile systemic amyloidosis (SSA); systemicfamilial amyloidosis; familial amyloidotic polyneuropathy (FAP);familial amyloidotic cardiomyopathy (FAC); and leptomeningealamyloidosis, also known as leptomeningeal or meningocerebrovascularamyloidosis, central nervous system (CNS) amyloidosis, or amyloidosisVII form.

In some embodiments of the methods of the invention, RNAi agents of theinvention are administered to subjects suffering from familialamyloidotic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA).Normal-sequence TTR causes cardiac amyloidosis in people who are elderlyand is termed senile systemic amyloidosis (SSA) (also called senilecardiac amyloidosis (SCA) or cardiac amyloidosis). SSA often isaccompanied by microscopic deposits in many other organs. TTR mutationsaccelerate the process of TTR amyloid formation and are the mostimportant risk factor for the development of clinically significant TTRamyloidosis (also called ATTR (amyloidosis-transthyretin type)). Morethan 85 amyloidogenic TTR variants are known to cause systemic familialamyloidosis.

In some embodiments of the methods of the invention, RNAi agents of theinvention are administered to subjects suffering from transthyretin(TTR)-related familial amyloidotic polyneuropathy (FAP). Such subjectsmay suffer from ocular manifestations, such as vitreous opacity andglaucoma. It is known to one of skill in the art that amyloidogenictransthyretin (ATTR) synthesized by retinal pigment epithelium (RPE)plays important roles in the progression of ocular amyloidosis. Previousstudies have shown that panretinal laser photocoagulation, which reducedthe RPE cells, prevented the progression of amyloid deposition in thevitreous, indicating that the effective suppression of ATTR expressionin RPE may become a novel therapy for ocular amyloidosis (see, e.g.,Kawaji, T., et al., Ophthalmology. (2010) 117: 552-555). The methods ofthe invention are useful for treatment of ocular manifestations of TTRrelated FAP, e.g., ocular amyloidosis. The RNAi agent can be deliveredin a manner suitable for targeting a particular tissue, such as the eye.Modes of ocular delivery include retrobulbar, subcutaneous eyelid,subconjunctival, subtenon, anterior chamber or intravitreous injection(or internal injection or infusion). Specific formulations for oculardelivery include eye drops or ointments.

Another TTR-associated disease is hyperthyroxinemia, also known as“dystransthyretinemic hyperthyroxinemia” or “dysprealbuminemichyperthyroxinemia”. This type of hyperthyroxinemia may be secondary toan increased association of thyroxine with TTR due to a mutant TTRmolecule with increased affinity for thyroxine. See, e.g., Moses et al.(1982) J. Clin. Invest., 86, 2025-2033.

The RNAi agents of the invention may be administered to a subject usingany mode of administration known in the art, including, but not limitedto subcutaneous, intravenous, intramuscular, intraocular,intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic,cerebrospinal, and any combinations thereof. In preferred embodiments,the agents are administered subcutaneously.

In some embodiments, the administration is via a depot injection. Adepot injection may release the RNAi agent in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof TTR, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the RNAi agent to the liver.

Other modes of administration include epidural, intracerebral,intracerebroventricular, nasal administration, intraarterial,intracardiac, intraosseous infusion, intrathecal, and intravitreal, andpulmonary. The mode of administration may be chosen based upon whetherlocal or systemic treatment is desired and based upon the area to betreated. The route and site of administration may be chosen to enhancetargeting.

In some embodiments, the RNAi agent is administered to a subject in anamount effective to inhibit TTR expression in a cell within the subject.The amount effective to inhibit TTR expression in a cell within asubject may be assessed using methods discussed above, including methodsthat involve assessment of the inhibition of TTR mRNA, TTR protein, orrelated variables, such as amyloid deposits.

In some embodiments, the RNAi agent is administered to a subject in atherapeutically or prophylactically effective amount.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a patientfor treating a TTR associated disease, is sufficient to effect treatmentof the disease (e.g., by diminishing, ameliorating or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, stage ofpathological processes mediated by TTR expression, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjectwho does not yet experience or display symptoms of a TTR-associateddisease, but who may be predisposed to the disease, is sufficient toprevent or ameliorate the disease or one or more symptoms of thedisease. Symptoms that may be ameliorated include sensory neuropathy(e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy(e.g., gastrointestinal dysfunction, such as gastric ulcer, ororthostatic hypotension), motor neuropathy, seizures, dementia,myelopathy, polyneuropathy, carpal tunnel syndrome, autonomicinsufficiency, cardiomyopathy, vitreous opacities, renal insufficiency,nephropathy, substantially reduced mBMI (modified Body Mass Index),cranial nerve dysfunction, and corneal lattice dystrophy. Amelioratingthe disease includes slowing the course of the disease or reducing theseverity of later-developing disease. The “prophylactically effectiveamount” may vary depending on the RNAi agent, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically-effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. RNAi agents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” also include an amount that providesa benefit in the treatment, prevention, or management of pathologicalprocesses or symptom(s) of pathological processes mediated by TTRexpression. Symptoms of TTR amyloidosis include sensory neuropathy (e.g.paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g.,gastrointestinal dysfunction, such as gastric ulcer, or orthostatichypotension), motor neuropathy, seizures, dementia, myelopathy,polyneuropathy, carpal tunnel syndrome, autonomic insufficiency,cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy,substantially reduced mBMI (modified Body Mass Index), cranial nervedysfunction, and corneal lattice dystrophy.

The dose of an RNAi agent that is administered to a subject may betailored to balance the risks and benefits of a particular dose, forexample, to achieve a desired level of TTR gene suppression (asassessed, e.g., based on TTR mRNA suppression, TTR protein expression,or a reduction in an amyloid deposit, as defined above) or a desiredtherapeutic or prophylactic effect, while at the same time avoidingundesirable side effects.

In one embodiment, the RNAi agent is administered at a dose of betweenabout 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg toabout 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, betweenabout 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg toabout 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg,between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg toabout 50 mg/kg.

In some embodiments, the RNAi agent is administered at a dose of about0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg,about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg,about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg,about 49 mg/kg or about 50 mg/kg.

In some embodiments, the RNAi agent is administered in two or moredoses. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable. In some embodiments, the number or amountof subsequent doses is dependent on the achievement of a desired effect,e.g., the suppression of a TTR gene, or the achievement of a therapeuticor prophylactic effect, e.g., reducing an amyloid deposit or reducing asymptom of a TTR-associated disease. In some embodiments, the RNAi agentis administered according to a schedule. For example, the RNAi agent maybe administered twice per week, three times per week, four times perweek, or five times per week. In some embodiments, the schedule involvesregularly spaced administrations, e.g., hourly, every four hours, everysix hours, every eight hours, every twelve hours, daily, every 2 days,every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly.In other embodiments, the schedule involves closely spacedadministrations followed by a longer period of time during which theagent is not administered. For example, the schedule may involve aninitial set of doses that are administered in a relatively short periodof time (e.g., about every 6 hours, about every 12 hours, about every 24hours, about every 48 hours, or about every 72 hours) followed by alonger time period (e.g., about 1 week, about 2 weeks, about 3 weeks,about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8weeks) during which the RNAi agent is not administered. In oneembodiment, the RNAi agent is initially administered hourly and is lateradministered at a longer interval (e.g., daily, weekly, biweekly, ormonthly). In another embodiment, the RNAi agent is initiallyadministered daily and is later administered at a longer interval (e.g.,weekly, biweekly, or monthly). In certain embodiments, the longerinterval increases over time or is determined based on the achievementof a desired effect. In a specific embodiment, the RNAi agent isadministered once daily during a first week, followed by weekly dosingstarting on the eighth day of administration. In another specificembodiment, the RNAi agent is administered every other day during afirst week followed by weekly dosing starting on the eighth day ofadministration.

Any of these schedules may optionally be repeated for one or moreiterations. The number of iterations may depend on the achievement of adesired effect, e.g., the suppression of a TTR gene, retinol bindingprotein level, vitamin A level, and/or the achievement of a therapeuticor prophylactic effect, e.g., reducing an amyloid deposit or reducing asymptom of a TTR-associated disease.

In some embodiments, the RNAi agent is administered with othertherapeutic agents or other therapeutic regimens. For example, otheragents or other therapeutic regimens suitable for treating aTTR-associated disease may include a liver transplant, which can reducemutant TTR levels in the body; Tafamidis (Vyndaqel), which kineticallystabilizes the TTR tetramer preventing tetramer dissociation requiredfor TTR amyloidogenesis; and diuretics, which may be employed, forexample, to reduce edema in TTR amyloidosis with cardiac involvement.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of an RNAi agent. The maintenance dose or dosescan be the same or lower than the initial dose, e.g., one-half of theinitial dose. A maintenance regimen can include treating the subjectwith a dose or doses ranging from 0.01 μg to 15 mg/kg of body weight perday, e.g., 10 mg/kg, 1 mg/kg, 0.1 mg/kg, 0.01 mg/kg, 0.001 mg/kg, or0.00001 mg/kg of bodyweight per day. The maintenance doses are, forexample, administered no more than once every 2 days, once every 5 days,once every 7 days, once every 10 days, once every 14 days, once every 21days, or once every 30 days. Further, the treatment regimen may last fora period of time which will vary depending upon the nature of theparticular disease, its severity and the overall condition of thepatient. In certain embodiments the dosage may be delivered no more thanonce per day, e.g., no more than once per 24, 36, 48, or more hours,e.g., no more than once every 5 or 8 days. Following treatment, thepatient can be monitored for changes in his/her condition. The dosage ofthe RNAi agent may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

VI. Kits

The present invention also provides kits for performing any of themethods of the invention. Such kits include one or more RNAi agent(s)and instructions for use, e.g., instructions for inhibiting expressionof a TTR in a cell by contacting the cell with the RNAi agent(s) in anamount effective to inhibit expression of the TTR. The kits mayoptionally further comprise means for contacting the cell with the RNAiagent (e.g., an injection device), or means for measuring the inhibitionof TTR (e.g., means for measuring the inhibition of TTR mRNA or TTRprotein). Such means for measuring the inhibition of TTR may comprise ameans for obtaining a sample from a subject, such as, e.g., a plasmasample. The kits of the invention may optionally further comprise meansfor administering the RNAi agent(s) to a subject or means fordetermining the therapeutically effective or prophylactically effectiveamount.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated herein by reference.

EXAMPLES Example 1: Inhibition of TTR with TTR-GalNAc Conjugates

A single dose of the TTR RNAi agent AD-43527 was administered to micesubcutaneously and TTR mRNA levels were determined 72 hours postadministration.

The mouse/rat cross-reactive GalNAc-conjugate, AD-43527, was chosen forin vivo evaluation in WT C57BL/6 mice for silencing of TTR mRNA inliver. The sequence of each strand of AD-43527 is shown below.

Strand: s=sense; an=antinense

Oligo Duplex # Strand # Sequence 5′to 3′ AD-43527 s A-AfaCfaGfuGfuUfcUfuGfcUfcUfaUfaAfL96 89592 (SEQ ID NO: 8) as A-uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu 83989 (SEQ ID NO: 9 L96 = GalNAc3;lowercase nts (a, u, g, c) are 2′-O-methyl nucleotides, Nf (i.e., Af) isa 2′-fluoro nucleotideThe ligand used was GalNAc₃:

This GalNAc3 ligand was conjugated to the 3′-end of the sense strandusing the linker and tether as shown below:

The structure of the resulting GalNAc₃ conjugated sense strand is shownin the following schematic:

Additional RNAi agents that target TTR and have the following sequencesand modifications were synthesized and assayed.

Mouse/rat cross reactive TTR RNAi agents Antisense strand DuplexSense strand 5′-3′ 5′-3′ AD- AfaCfaGfuGfuUfcUfuG uUfaUfaGfaGfcAfaGf43528 fcUfcUfaUfaAfQ11L96 aAfcAfcUfgUfusUfsu (SEQ ID NO: 10)(SEQ ID NO: 11)Human/cyno cross reactive TTR RNAi agents; parent duplex is AD-18328[having a sense strand 5′-3′ sequence of GuAAccAAGAGuAuuccAudTdT (SEQ IDNO: 12) and antisense strand 5′ to 3′ sequence ofAUGGAAuACUCUUGGUuACdTdT (SEQ ID NO: 13) with the followingmodifications: alternating 2′F/2′OMe w/2 PS on AS.

Duplex Sense strand 5′-3′ Antisense strand 5′-3′ AD- AfuGfuAfaCfcAfaGfaGaUfgGfaAfuAfcUfcUfuGf 45163 fuAfuUfcCfaUfL96 gUfuAfcAfusGfsa(SEQ ID NO: 14) (SEQ ID NO: 16) AD- AfuGfuAfaCfcAfaGfaGaUfgGfaAfuAfcUfcUfuGf 45164 fuAfuUfcCfaUfQ11L96 gUfuAfcAfusGfsa(SEQ ID NO: 15) (SEQ ID NO: 17) L96 = GalNAc₃; lowercase nts (a, u, g,c) are 2′-O-methyl nucleotides, Nf (i.e., Af) is a 2′-fluoro nucleotide;Q11 is cholesterol; s is phosphorothioate.

AD-43527 was administered to female C57BL6 mice (6-10 weeks, 5 pergroup) via subcutaneous injection at a dose volume of 10 μl/g at a doseof 30, 15, 7.5, 3.5, 1.75 or 0.5 mg/kg of AD-43527. Control animalsreceived PBS by subcutaneous injection at the same dose volume.

After approximately seventy two hours, mice were anesthetized with 200μl of ketamine, and then exsanguinated by severing the right caudalartery. Liver tissue was collected, flash-frozen and stored at −80° C.until processing.

Efficacy of treatment was evaluated by measurement of TTR mRNA in theliver at 72 hours post-dose. TTR liver mRNA levels were assayedutilizing the Branched DNA assays-QuantiGene 1.0 (Panomics). Briefly,mouse liver samples were ground and tissue lysates were prepared. Liverlysis mixture (a mixture of 1 volume of lysis mixture, 2 volume ofnuclease-free water and 10 μl of Proteinase-K/ml for a finalconcentration of 20 mg/ml) was incubated at 65° C. for 35 minutes. 5 μlof liver lysate and 95 μl of working probe set (TTR probe for genetarget and GAPDH for endogenous control) were added into the CapturePlate. Capture Plates were incubated at 53° C.±1° C. (aprx. 16-20 hrs).The next day, the Capture Plates were washed 3 times with 1× Wash Buffer(nuclease-free water, Buffer Component 1 and Wash Buffer Component 2),then dried by centrifuging for 1 minute at 240 g. 100 μl of AmplifierProbe mix per well was added into the Capture Plate, which was sealedwith aluminum foil and incubated for 1 hour at 46° C.±1° C. Following a1 hour incubation, the wash step was repeated, then 100 μl of LabelProbe mix per well was added. Capture plates were incubated at 46° C.±1°C. for 1 hour. The plates were then washed with 1× Wash Buffer, driedand 100 μl substrate per well was added into the Capture Plates. CapturePlates were incubated for 30 minutes at 46° C. followed by incubationfor 30 minutes at room temperature. Plates were read using theSpectraMax Luminometer following incubation. bDNA data were analyzed bysubtracting the average background from each duplicate sample, averagingthe resultant duplicate GAPDH (control probe) and TTR (experimentalprobe) values, and then computing the ratio: (experimentalprobe-background)/(control probe-background). The average TTR mRNA levelwas calculated for each group and normalized to the PBS group average togive relative TTR mRNA as a % of the PBS control group.

The results are shown in FIG. 1 . The GalNAc conjugated RNAi agenttargeting TTR had an ED₅₀ of approximately 5 mg/kg for TTR mRNAknockdown. These results demonstrate that GalNAc conjugated RNAi agentsthat target TTR are effective at inhibiting expression of TTR mRNA.

Example 2: Inhibition of TTR with TTR-GalNAc Conjugates is Durable

Mice were administered a subcutaneous dose (either 7.5 or 30.0 mg/kg) ofAD-43527, a GalNAc conjugated RNAi agent that targets TTR. The TTR mRNAlevels in the liver were evaluated at 1, 3, 5, 7, 10, 13, 15, 19, 26,33, and 41 days post treatment using the method described in Example 1.

The results are shown in FIG. 2 . At day 19, administration of 30.0mg/kg GalNAc conjugated RNAi agents still showed about 50% silencing.Full recovery of expression occurred at day 41.

These results demonstrated that the inhibition provided by GalNAcconjugated siRNA targeting TTR is durable, lasting up to 3, 5, 7, 10,13, 15, 19, 26 or 33 days post treatment.

Example 3. RNA Synthesis and Duplex Annealing 1. OligonucleotideSynthesis

Oligonucleotides were synthesized on an AKTAoligopilot synthesizer or anABI 394 synthesizer. Commercially available controlled pore glass solidsupport (dT-CPG, 500{acute over (Å)}, Prime Synthesis) and RNAphosphoramidites with standard protecting groups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2--isobutyryl-2′-t-butyldimethylsilyl-guanosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis unless otherwise specified. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using a solid supportcontaining the corresponding ligand. For example, the introduction of acarbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of asequence was achieved by starting the synthesis with the correspondingcarbohydrate solid support. Similarly a cholesterol moiety at the 3′-endwas introduced by starting the synthesis on the cholesterol support. Ingeneral, the ligand moiety was tethered to trans-4-hydroxyprolinol via atether of choice as described in the previous examples to obtain ahydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety wasthen coupled to a solid support via a succinate linker or was convertedto phosphoramidite via standard phosphitylation conditions to obtain thedesired carbohydrate conjugate building blocks. Fluorophore labeledsiRNAs were synthesized from the corresponding phosphoramidite or solidsupport, purchased from Biosearch Technologies. The oleyl lithocholic(GalNAc)₃ polymer support made in house at a loading of 38.6 μmol/gram.The Mannose (Man)₃ polymer support was also made in house at a loadingof 42.0 μmol/gram.

Conjugation of the ligand of choice at the desired position, for exampleat the 5′-end of the sequence, was achieved by coupling of thecorresponding phosphoramidite to the growing chain under standardphosphoramidite coupling conditions unless otherwise specified. Anextended 15 minute coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported in Beaucage, S. L. (2008) Solid-phase synthesis of siRNAoligonucleotides. Curr. Opin. Drug Discov. Devel., 11, 203-216; Mueller,S., Wolf, J. and Ivanov, S. A. (2004) Current Strategies for theSynthesis of RNA. Curr. Org. Synth., 1, 293-307; Xia, J., Noronha, A.,Toudjarska, I., Li, F., Akinc, A., Braich, R., Frank-Kamenetsky, M.,Rajeev, K. G., Egli, M. and Manoharan, M. (2006) Gene Silencing Activityof siRNAs with a Ribo-difluorotoluyl Nucleotide. ACS Chem. Biol., 1,176-183 or by treatment with tert-butyl hydroperoxide/acetonitrile/water(10:87:3) with a 10 minute oxidation wait time conjugatedoligonucleotide. Phosphorothioate was introduced by the oxidation ofphosphite to phosphorothioate by using a sulfur transfer reagent such asDDTT (purchased from AM Chemicals), PADS and or Beaucage reagent Thecholesterol phosphoramidite was synthesized in house, and used at aconcentration of 0.1 M in dichloromethane. Coupling time for thecholesterol phosphoramidite was 16 minutes.

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 80 mLof a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at55° C. The bottle was cooled briefly on ice and then the ethanolicammonia mixture was filtered into a new 250 ml bottle. The CPG waswashed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume ofthe mixture was then reduced to ˜30 ml by roto-vap. The mixture was thenfrozen on dry ice and dried under vacuum on a speed vac.

3. Deprotection-H (Removal of 2′ TBDMS Group)

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

4. Analysis

The oligonucleotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified by reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides werediluted in water to 150 μl and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. RNAi Agent Preparation

For the preparation of an RNAi agent, equimolar amounts of sense andantisense strand were heated in 1×PBS at 95° C. for 5 minutes and slowlycooled to room temperature. The integrity of the duplex was confirmed byHPLC analysis. Table 1 below reflects the RNAi agents which target humanor rodent TTR mRNA.

TABLE 1 RNAi Agents and Results of In Vitro Screening SEQ SEQ% of mRNA remained Duplex ID ID conc. of siRNA IC50 ID S ID NO:Sense strand (S) AS ID NO: Antisense strand (AS) 1 nM 0.1 nM 0.01 nM(nM) D1000 S1000 18 AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasu AS1000 1110AfUfgGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.1 0.47 0.006 D1001 S1001 19AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1001 1111aUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.10 0.49 0.0065 D1002 S100220 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1002 1112aUfgGfAfAfuAfcUfcuuGfgsUfuAfcAfusGfsa 0.04 0.10 0.46 0.0068 D1003 S100321 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1003 1113aUfgGfAfAfuAfcUfcuuGfgUfsuAfcAfusGfsa 0.05 0.12 0.56 0.0073 D1004 S100422 aUGuaACccAGagUAuuCCasu AS1004 1114 AUggAAuaCUcuUGguUAcaUsGsa 0.070.13 0.44 0.008 D1005 S1005 23 AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS10051115 aUfgGfAfAfuAfcUfcuuGfgsUfsuAfcAfusGfsa 0.06 0.11 0.53 0.0093 D1006S1006 24 AfuGfuAfAfccAfAfGfaGfuAfuUfcCfasUf AS1006 1116aUfgGfaAfuAfcUfcuuGfGfuuAfcAfusGfsa 0.05 0.16 0.55 0.0095 D1007 S1007 25AfuGfuAfAfCfcAfAfGfaGfuAfuUfcCfasUf AS1007 1117aUfgGfaAfuAfcUfcuuGfguuAfcAfusGfsa 0.05 0.14 0.48 0.0098 D1008 S1008 26auguaaccaadGadGudAudAcdGasu AS1008 1118aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa 0.07 0.11 0.33 0.010 D1009 S1009 27UfgGfGfAfuUfuCfAfUfgUfaAfcCfAfAfgsAf AS1009 1119uCfuugGfuUfaCfaugAfaAfuccCfasUfsc 0.03 0.14 0.56 0.0101 D1010 S1010 28UfgGfgauUfuCfAfUfgUfaAfcCfaAfgsAf AS1010 1120uCfuUfgGfuUfaCfaugAfaAfUfCfcCfasUfsc 0.03 0.14 0.65 0.0101 D1011 S101129 aUfGfuAfAfccAfAfGfaGfuAfuUfcCfasUf AS1011 1121aUfgGfaAfuAfcUfcuuGfGfuuAfcaUfsgsa 0.06 0.10 0.55 0.011 D1012 S1012 30UfgGfgAfuUfuCfAfUfgUfaacCfaAfgsAf AS1012 1122uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc 0.04 0.13 0.54 0.0114 D1013 S101331 auguaaccaadGadGudAudAcdGasu AS1013 1123aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa 0.11 0.19 0.49 0.011 D1014 S1014 32AfuGfuaaCfcAfAfGfaGfuAfuUfcCfasUf AS1014 1124aUfgGfaAfuAfcUfcuuGfgUfUfAfcAfusGfsa 0.04 0.16 0.59 0.013 D1015 S1015 33AfuguAfaccAfaGfdAGfdTAdTudCcdAsu AS1015 1125dAUdGgdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa 0.07 0.15 0.51 0.013 D1016 S101634 auGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1016 1126aUfgGfaAfuAfcUfcuuGfgUfuAfcAfUfsGfsa 0.05 0.14 0.64 0.013 D1017 S1017 35UfGfggAfuUfuCfAfUfgUfAfAfcCfaAfgsAf AS1017 1127uCfuUfgGfuuaCfaugAfaAfuCfCfcasUfsc 0.09 0.41 0.74 0.0133 D1018 S1018 36AfuguAfaCfcAfAfGfaGfuAfuUfcCfasUf AS1018 1128aUfgGfaAfuAfcUfcuuGfgUfuAfCfAfusGfsa 0.03 0.14 0.61 0.014 D1019 S1019 37AfuGfuAfaccAfAfGfaGfuAfuUfcCfasUf AS1019 1129aUfgGfaAfuAfcUfcuuGfGfUfuAfcAfusGfsa 0.02 0.2 0.7 0.014 D1020 S1020 38AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1020 1130asUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.04 0.16 0.67 0.0156 D1021 S102139 aUfguAfAfccAfAfgagUfaUfuCfcasUf AS1021 1131aUfGfgAfaUfaCfUfCfuuGfGfuuAfCfaUfsgsa 0.11 0.24 0.64 0.016 D1022 S102240 dTdGggdAdTuudCdAugdTdAacdCdAagsdA AS1022 1132udCdTugdGdTuadCdAugdAdAaudCdCcasdTsc 0.08 0.27 0.64 0.0161 D1023 S102341 AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf AS1023 1133aUfgsGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa 0.03 0.19 0.63 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1065GfaUfcUfgCfaGfCfCfaUfuAfAfAfaAfgAfL96 AS2047 2157uCfuUfuuuAfaUfggcUfgCfaGfaUfcsAfsu D2048 S2048 1066AfuCfuGfcAfgCfCfAfuUfaaaAfaGfaCfL96 AS2048 2158gUfcUfuUfUfUfaAfuggCfuGfcAfgAfusCfsa D2049 S2049 1067AfuCfuGfcAfgCfCfAfuUfaAfAfAfaGfaCfL96 AS2049 2159gUfcUfuuuUfaAfuggCfuGfcAfgAfusCfsa D2050 S2050 1068UfcUfgCfaGfcCfAfUfuAfaaaAfgAfcAfL96 AS2050 2160uGfuCfuUfUfUfuAfaugGfcUfgCfaGfasUfsc D2051 S2051 1069UfcUfgCfaGfcCfAfUfuAfaAfAfAfgAfcAfL96 AS2051 2161uGfuCfuuuUfuAfaugGfcUfgCfaGfasUfsc D2052 S2052 1070CfuGfcAfgCfcAfUfUfaAfaaaGfaCfaCfL96 AS2052 2162gUfgUfcUfUfUfuUfaauGfgCfuGfcAfgsAfsu D2053 S2053 1071CfuGfcAfgCfcAfUfUfaAfaAfAfGfaCfaCfL96 AS2053 2163gUfgUfcuuUfuUfaauGfgCfuGfcAfgsAfsu D2054 S2054 1072UfgCfaGfcCfaUfUfAfaAfaagAfcAfcAfL96 AS2054 2164uGfuGfuCfUfUfuUfuaaUfgGfcUfgCfasGfsa D2055 S2055 1073UfgCfaGfcCfaUfUfAfaAfaAfGfAfcAfcAfL96 AS2055 2165uGfuGfucuUfuUfuaaUfgGfcUfgCfasGfsa D2056 S2056 1074GfcAfgCfcAfuUfAfAfaAfagaCfaCfaUfL96 AS2056 2166aUfgUfgUfCfUfuUfuuaAfuGfgCfuGfcsAfsg D2057 S2057 1075GfcAfgCfcAfuUfAfAfaAfaGfAfCfaCfaUfL96 AS2057 2167aUfgUfgucUfuUfuuaAfuGfgCfuGfcsAfsg D2058 S2058 1076CfaGfcCfaUfuAfAfAfaAfgacAfcAfuUfL96 AS2058 2168aAfuGfuGfUfCfuUfuuuAfaUfgGfcUfgsCfsa D2059 S2059 1077CfaGfcCfaUfuAfAfAfaAfgAfCfAfcAfuUfL96 AS2059 2169aAfuGfuguCfuUfuuuAfaUfgGfcUfgsCfsa D2060 S2060 1078AfgCfcAfuUfaAfAfAfaGfacaCfaUfuCfL96 AS2060 2170gAfaUfgUfGfUfcUfuuuUfaAfuGfgCfusGfsc D2061 S2061 1079AfgCfcAfuUfaAfAfAfaGfaCfAfCfaUfuCfL96 AS2061 2171gAfaUfgugUfcUfuuuUfaAfuGfgCfusGfsc D2062 S2062 1080GfcCfaUfuAfaAfAfAfgAfcacAfuUfcUfL96 AS2062 2172aGfaAfuGfUfGfuCfuuuUfuAfaUfgGfcsUfsg D2063 S2063 1081GfcCfaUfuAfaAfAfAfgAfcAfCfAfuUfcUfL96 AS2063 2173aGfaAfuguGfuCfu uuUfuAfaUfgGfcsUfsg D2064 S2064 1082CfcAfuUfaAfaAfAfGfaCfacaUfuCfuGfL96 AS2064 2174cAfgAfaUfGfUfgUfcuuUfuUfaAfuGfgsCfsu D2065 S2065 1083CfcAf uUfaAfaAfAfGfaCfaCfAfUfuCfuGfL96 AS2065 2175cAfgAfaugUfgUfcuuUfuUfaAfuGfgsCfsu D2066 S2066 1084CfaUfuAfaAfaAfGfAfcAfcauUfcUfgUfL96 AS2066 2176aCfaGfaAfUfGfuGfucuUfuUfuAfaUfgsGfsc D2067 S2067 1085CfaUfuAfaAfaAfGfAfcAfcAfUfUfcUfgUfL96 AS2067 2177aCfaGfaauGfuGfucuUfuUfuAfaUfgsGfsc D2068 S2068 1086AfuUfaAfaAfaGfAfCfaCfauuCfuGfuAfL96 AS2068 2178uAfcAfgAfAfUfgUfgucUfuUfuUfaAfusGfsg D2069 S2069 1087AfuUfaAfaAfaGfAfCfaCfaUfUfCfuGfuAfL96 AS2069 2179uAfcAfgaaUfgUfgucUfuUfuUfaAfusGfsg D2070 S2070 1088UfuAfaAfaAfgAfCfAfcAfuucUfgUfaAfL96 AS2070 2180uUfaCfaGfAfAfuGfuguCfuUfuUfuAfasUfsg D2071 S2071 1089UfuAfaAfaAfgAfCfAfcAfuUfCfUfgUfaAfL96 AS2071 2181uUfaCfagaAfuGfuguCfuUfuUfuAfasUfsg D2072 S2072 1090UfaAfaAfaGfaCfAfCfaUfucuGfuAfaAfL96 AS2072 2182uUfuAfcAfGfAfaUfgugUfcUfuUfuUfasAfsu D2073 S2073 1091UfaAfaAfaGfaCfAfCfaUfuCfUfGfuAfaAfL96 AS2073 2183uUfuAfcagAfaUfgugUfcUfuUfuUfasAfsu D2074 S2074 1092AfaAfaAfgAfcAfCfAfuUfcugUfaAfaAfL96 AS2074 2184uUfuUfaCfAfGfaAfuguGfuCfuUfuUfusAfsa D2075 S2075 1093AfaAfaAfgAfcAfCfAfuUfcUfGfUfaAfaAfL96 AS2075 2185uUfuUfacaGfaAfuguGfuCfuUfuUfusAfsa D2076 S2076 1094AfaAfaGfaCfaCfAfUfuCfuguAfaAfaAfL96 AS2076 2186uUfuUfuAfCfAfgAfaugUfgUfcUfuUfusUfsa D2077 S2077 1095AfaAfaGfaCfaCfAfUfuCfuGfUfAfaAfaAfL96 AS2077 2187uUfuUfuacAfgAfaugUfgUfcUfuUfusUfsa D2078 S2078 1096AfaAfgAfcAfcAfUfUfcUfguaAfaAfaAfL96 AS2078 2188uUfuUfuUfAfCfaGfaauGfuGfuCfuUfusUfsu D2079 S2079 1097AfaAfgAfcAfcAfUfUfcUfgUfAfAfaAfaAfL96 AS2079 2189uUfuUfuuaCfaGfaauGfuGfuCfuUfusUfsu D2080 S2080 1098AfaGfaCfaCfaUfUfCfuGfuaaAfaAfaAfL96 AS2080 2190uUfuUfuUfUfAfcAfgaaUfgUfgUfcUfusUfsu D2081 S2081 1099AfaGfaCfaCfaUfUfCfuGfuAfAfAfaAfaAfL96 AS2081 2191uUfuUfuuuAfcAfgaaUfgUfgUfcUfusUfsu D2082 S2082 1100AfgAfcAfcAfuUfCfUfgUfaaaAfaAfaAfL96 AS2082 2192uUfuUfuUfUfUfaCfagaAfuGfuGfuCfusUfsu D2083 S2083 1101AfgAfcAfcAfuUfCfUfgUfaAfAfAfaAfaAfL96 AS2083 2193uUfuUfuuuUfaCfagaAfuGfuGfuCfusUfsu D2084 S2084 1102GfaCfaCfaUfuCfUfGfuAfaaaAfaAfaAfL96 AS2084 2194uUfuUfuUfUfUfuAfcagAfaUfgUfgUfcsUfsu D2085 S2085 1103GfaCfaCfaUfuCfUfGfuAfaAfAfAfaAfaAfL96 AS2085 2195uUfuUfuuuUfuAfcagAfaUfgUfgUfcsUfsu D2086 S2086 1104AfcAfcAfuUfcUfGfUfaAfaaaAfaAfaAfL96 AS2086 2196uUfuUfuUfUfUfuUfacaGfaAfuGfuGfusCfsu D2087 S2087 1105AfcAfcAfuUfcUfGfUfaAfaAfAfAfaAfaAfL96 AS2087 2197uUfuUfuuuUfuUfacaGfaAfuGfuGfusCfsu D2088 S2088 1106CfaCfaUfuCfuGfUfAfaAfaaaAfaAfaAfL96 AS2088 2198uUfuUfuUfUfUfuUfuacAfgAfaUfgUfgsUfsc D2089 S2089 1107CfaCfaUfuCfuGfUfAfaAfaAfAfAfaAfaAfL96 AS2089 2199uUfuUfuuuUfuUfuacAfgAfaUfgUfgsUfsc D2090 S2090 1108AfcAfuUfcUfgUfAfAfaAfaaaAfaAfaAfL96 AS2090 2200uUfuUfuUfUfUfuUfuuaCfaGfaAfuGfusGfsu D2091 S2091 1109AfcAfuUfcUfgUfAfAfaAfaAfAfAfaAfaAfL96 AS2091 2201uUfuUfuuuUfuUfuuaCfaGfaAfuGfusGfsu Lowercase nucleotides (a, u, g, c)are 2′-O-methyl nucleotides; Nf (e.g., Af) is a 2′-fluoro nucleotide; sis a phosphothiorate linkage; L96 indicates a GalNAc₃ ligand.

Example 4: In Vitro Screening of RNAi Agents Cell Culture andTransfections

Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, Va.) were grownto near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Transfection wascarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl ofsiRNA duplexes per well into a 96-well plate and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotic containing ˜2×104 Hep3B cells were then added to the siRNAmixture. Cells were incubated for either 24 or 120 hours prior to RNApurification. Single dose experiments were performed at 10 nM and 0.1 nMfinal duplex concentration and dose response experiments were done using8, 4 fold serial dilutions with a maximum dose of 10 nM final duplexconcentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen. Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed was the same throughout the process). Ten microliters ofmagnetic beads and 80 μl Lysis/Binding Buffer mixture were added to around bottom plate and mixed for 1 minute. Magnetic beads were capturedusing magnetic stand and the supernatant was removed without disturbingthe beads. After removing the supernatant, the lysed cells were added tothe remaining beads and mixed for 5 minutes. After removing thesupernatant, magnetic beads were washed 2 times with 150 μl Wash BufferA and mixed for 1 minute. Beads were capture again and supernatantremoved. Beads were then washed with 150 μl Wash Buffer B, captured andsupernatant was removed. Beads were next washed with 150 μl ElutionBuffer, captured and supernatant removed. Beads were allowed to dry for2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μlof supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, CALIF., Cat #4368813)

A master mix of 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl Random primers,0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H₂Oper reaction were added into 5 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)),0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914_ml (human)cat #Rn00562124_ml (rat)) and 5 μl Lightcycler 480 probe master mix(Roche Cat #04887301001) per well in a 384 well plate (Roche cat#04887301001). Real time PCR was done in a Roche LC 480 Real Time PCRmachine (Roche). Each duplex was tested in at least two independenttransfections and each transfection was assayed in duplicate, unlessotherwise noted.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 (sense sequence: cuuAcGcuGAGuAcuucGAdTsdT(SEQ ID NO: 2202); antisense sequence: UCGAAGuCUcAGCGuAAGdTsdT (SEQ IDNO: 2203)) or naïve cells over the same dose range, or to its own lowestdose. IC₅₀s were calculated for each individual transfection as well asin combination, where a single IC₅₀ was fit to the data from bothtransfections.

The results of gene silencing of the exemplary siRNA duplex with variousmotif modifications of the invention are shown in Table 1 above.

Example 5: In Vitro Silencing Activity of Chemically Modified RNAiAgents that Target TTR

The following experiments demonstrated the beneficial effects ofchemical modifications, including the introduction of triplet repeatmotifs, together with a GalNAc₃ ligand, on the silencing activity ofRNAi agents that target TTR. The sequences of the agents investigatedare provided in Table 2 below. The regions of complementarity to the TTRmRNA are as follows: the region of complementarity of RNAi agentsAD-45165, AD-51546 and AD-51547 is GGATGGGATTTCATGTAACCAAGA (SEQ ID NO:2204) and the region or complementarity of RNAi agents AD-45163,AD-51544, and AD-51545 is TTCATGTAACCAAGAGTATTCCAT (SEQ ID NO: 2205).

Protocol for Assessment of IC₅₀ in Hep3B Cells

The IC₅₀ for each modified siRNA was determined in Hep3B cells (a humanhepatoma cell line) by standard reverse transfection using LipofectamineRNAiMAX. In brief, reverse transfection was carried out by adding 5 μLof Opti-MEM to 5 μL of siRNA duplex per well into a 96-well plate alongwith 10 μL of Opti-MEM plus 0.5 μL of Lipofectamine RNAiMax per well(Invitrogen, Carlsbad Calif. cat #13778-150) and incubating at roomtemperature for 15-20 minutes. Following incubation, 100 μL of completegrowth media without antibiotic containing 12,000-15,000 Hep3B cells wasthen added to each well. Cells were incubated for 24 hours at 37° C. inan atmosphere of 5% CO₂ prior to lysis and analysis of TTR and GAPDHmRNA by bDNA (Quantigene). Seven different siRNA concentrations rangingfrom 10 nM to 0.6 μM were assessed for IC₅₀ determination and TTR/GAPDHfor siRNA transfected cells was normalized to cells transfected with 10nM Luc siRNA. The results are shown in Table 2.

Protocol for Assessment of Free-Uptake IC₅₀

Free uptake silencing in primary cynomolgus hepatocytes was assessedfollowing incubation with TTR siRNA for either 4 hours or 24 hours.Silencing was measured at 24 hours from the initial exposure. In brief,96-well culture plates were coated with 0.05%-0.1% collagen (SigmaC3867-1VL) at room temperature, 24 hours prior to the start of theexperiment. On the day of assay, siRNAs were diluted in pre-warmedPlating Media consisting of DMEM supplemented with GIBCO's MaintenanceMedia Kit (Serum-Free, Life Technologies CM4000), and added to thecollagen-coated 96-well culture plates. Cryopreserved primary cynomolgushepatocytes were rapidly thawed in a 37° C. water bath, and immediatelydiluted in Plating Media to a concentration of 360,000 cells/mL. Avolume of cell suspension was gently pipetted on top of the pre-platedsiRNAs such that the final cell count was 18,000 cells/well. The platewas lightly swirled to mix and spread cells evenly across the wells andplaced in a 37° C., 5% CO₂ incubator for 24 hours prior to lysis andanalysis of TTR and GAPDH mRNA by bDNA (Quantigene, Affymetrix). In thecase of the 4 h incubation with siRNA, the media was decanted after 4hours of exposure to the cells, and replaced with fresh Plating Mediafor the remaining 20 hours of incubation. Downstream analysis for TTRand GAPDH mRNA was the same as described above. For a typical doseresponse curve, siRNAs were titrated from 1 uM to 0.24 nM by 4 foldserial dilution.

TABLE 2In vitro Activity Summary for Alternating TTR-GaINAc and Variants with Triplet MotifsFree-Uptake Hep3B IC50 (μM) IC50 Duplex ID S (5′-3′) AS (5′-3″) 4 h 24 h(nM) AD-45163 AfuGfuAfaCfcAfaGfaGfuAfuUfc aUfgGfaAfuAfcUfcUfuGfgUfuAfc0.04101 0.00820 0.0115 CfaUfL96 (SEQ ID NO: 2206)AfusGfsa (SEQ ID NO: 2212) AD-51544 AfuGfuAfaCfcAfAfGfaGfuAfuucaUfgGfAfAfuAfcUfcuuGfgUfuAfc 0.00346 0.00374 0.0014CfaUfL96 (SEQ ID NO: 2207) AfusGfsa (SEQ ID NO: 2213) AD-51545AfuGfuAfAfCfcAfAfGfaGfuAfuU aUfgGfaAfuAfcUfcuuGfguuAfcA 0.00395 0.003890.0018 fcCfaUfL96 (SEQ ID NO: 2208) fusGfsa (SEQ ID NO: 2214) AD-45165UfgGfgAfuUfuCfaUfgUfaAfcCfa uCfuUfgGfuUfaCfaUfgAfaAfuCf 0.02407 0.008690.0112 AfgAfL96 (SEQ ID NO: 2209) cCfasUfsc (SEQ ID NO: 2215) AD-51546UfgGfGfAfuUfuCfAfUfgUfaAfcC uCfuugGfuUfaCfaugAfaAfuccC 0.00317 0.002630.0017 fAfAfgAfL96 (SEQ ID NO: 2210) fasUfsc (SEQ ID NO: 2216) AD-51547UfgGfgAfuUfuCfAfUfgUfaacCfa uCfuUfgGfUfUfaCfaugAfaAfuC 0.00460 0.003740.0028 AfgAfL96 (SEQ ID NO: 2211) fcCfasUfsc (SEQ ID NO: 2217) Lowercasenucleotides (a, u, g, c) indicate 2′-O-methyl nucleotides; Nf (e.g., Af)indicates a 2′-fluoro nucleotide; s indicates a phosphothiorate linkage;L96 indicates a GalNAc3 ligand; bold nucleotides indicate changesrelative to the corresponding parent agent. Each bold nucleotide is atthe center of a triplet motif.

The results are provided in Table 2 and demonstrate that modified RNAiagents that target TTR provide enhanced silencing activity.

Results: Improved Activity of Modified RNAi Agents

Parent RNAi agents with alternating chemical modifications and a GalNAc₃ligand provided an IC₅₀ in Hep3B cells of about 0.01 nM. As shown inFIGS. 4-5 and in Table 2, agents modified relative to the parent agents,for example, by the addition of one or more repeating triplets of2′-fluoro and 2′-O-methyl modifications, showed unexpectedly enhancedsilencing activity, achieving IC₅₀ values in Hep3B cells that were 5-8fold better than the corresponding parent agent.

Results: Free Uptake IC₅₀s in Hep3B Cells

As shown in Table 2 and FIGS. 6-7 , RNAi agents modified relative to theparent AD-45163 also showed enhanced free uptake silencing. The modifiedagents showed more than double the silencing activity of the parentafter a 24 hour incubation period and nearly 10 times the silencingactivity of the parent after a 4 hour incubation period.

As shown in Table 2 and FIGS. 8-9 , RNAi agents modified relative to theparent AD-45165 also showed enhanced free uptake silencing. The modifiedagents showed 2-3 times the silencing activity of the parent after a 24hour incubation period and 5-8 times the silencing activity of theparent after a 4 hour incubation period.

Taken collectively, these results demonstrate that the modified RNAiagents presented herein, e.g., AD-51544, AD-51545, AD-51546, andAD-51547, all showed unexpectedly good inhibition of TTR mRNA in invitro silencing experiments.

Example 6: TTR mRNA Silencing and TTR Protein Suppression in TransgenicMice

To assess the efficacy of the RNAi agents AD-45163, AD-51544, AD-51545,AD45165, AD-51546, and AD-51547, these agents were administered totransgenic mice that express human transthyretin with the V30M mutation(see Santos, S D., Fernaandes, R., and Saraiva, M J. (2010) Neurobiologyof Aging, 31, 280-289). The V30M mutation is known to cause familialamyloid polyneuropathy type I in humans. See, e.g., Lobato, L. (2003) JNephrol., 16(3):438-42.

The RNAi agents (in PBS buffer) or PBS control were administered to mice(2 male and 2 female) of 18-24 months of age in a single subcutaneousdose of 5 mg/kg or 1 mg/kg. After approximately 48 hours, mice wereanesthetized with 200 μl of ketamine, and then exsanguinated by severingthe right caudal artery. Whole blood was isolated and plasma wasisolated and stored at −80° C. until assaying. Liver tissue wascollected, flash-frozen and stored at −80° C. until processing.

Efficacy of treatment was evaluated by (i) measurement of TTR mRNA inliver at 48 hours post-dose, and (ii) measurement of TTR protein inplasma at pre-bleed and at 48 hours post-dose. TTR liver mRNA levelswere assayed utilizing the Branched DNA assays-QuantiGene 2.0 (Panomicscat #: QS0011). Briefly, mouse liver samples were ground and tissuelysates were prepared. Liver lysis mixture (a mixture of 1 volume oflysis mixture, 2 volume of nuclease-free water and 10 ul ofProteinase-K/ml for a final concentration of 20 mg/ml) was incubated at65° C. for 35 minutes. 20 μl of Working Probe Set (TTR probe for genetarget and GAPDH for endogenous control) and 80 ul of tissue-lysate werethen added into the Capture Plate. Capture Plates were incubated at 55°C.±1° C. (aprx. 16-20 hrs). The next day, the Capture Plates were washed3 times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 andWash Buffer Component 2), then dried by centrifuging for 1 minute at 240g. 100 μl of pre-Amplifier Working Reagent was added into the CapturePlate, which was sealed with aluminum foil and incubated for 1 hour at55° C.±1° C. Following 1 hour incubation, the wash step was repeated,then 100 μl of Amplifier Working Reagent was added. After 1 hour, thewash and dry steps were repeated, and 100 μl of Label Probe was added.Capture plates were incubated 50° C.±1° C. for 1 hour. The plate wasthen washed with 1× Wash Buffer, dried and 100 μl Substrate was addedinto the Capture Plate. Capture Plates were read using the SpectraMaxLuminometer following a 5 to 15 minute incubation. bDNA data wereanalyzed by subtracting the average background from each triplicatesample, averaging the resultant triplicate GAPDH (control probe) and TTR(experimental probe) values, and then computing the ratio: (experimentalprobe-background)/(control probe-background).

Plasma TTR levels were assayed utilizing the commercially available kit“AssayMax Human Prealbumin ELISA Kit” (AssayPro, St. Charles, Mo.,Catalog #EP3010-1) according to manufacturer's guidelines. Briefly,mouse plasma was diluted 1:10,000 in 1× mix diluents and added topre-coated plates along with kit standards, and incubated for 2 hours atroom temperature followed by 5× washes with kit wash buffer. Fiftymicroliters of biotinylated prealbumin antibody was added to each welland incubated for 1 hr at room temperature, followed by 5× washes withwash buffer. Fifty microliters of streptavidin-peroxidase conjugate wasadded to each well and plates were incubated for 30 minutes at roomtemperature followed by washing as previously described. The reactionwas developed by the addition of 50 μl/well of chromogen substrate andincubation for 10 minutes at room temperature with stopping of reactionby the addition of 50 μl/well of stop solution. Absorbance at 450 nm wasread on a Versamax microplate reader (Molecular Devices, Sunnyvale,Calif.) and data were analyzed utilizing the Softmax 4.6 softwarepackage (Molecular Devices).

The results are shown in FIGS. 10-12 . FIGS. 10A and 10B show that theRNAi agents modified relative to the parent agents AD-45163 and AD-45165showed RNA silencing activity that was similar or more potent comparedwith that of the parent agents. FIG. 11 shows that the agents AD-51544and AD-51545 showed dose dependent silencing activity and that thesilencing activity of these agents at a dose of 5 mg/kg was similar tothat of the corresponding parent AD-45163. FIG. 12 shows that the agentsAD-51546 and AD-51547 also showed dose-dependent silencing activity.Furthermore, the silencing activity of AD-51546 and AD-51547 at a doseof 5 mg/kg was superior to that of the corresponding parent AD-45165.

Example 7: Serum and Liver Pharmacokinetic Profiles of RNAi Agents thatTarget TTR in Mice

To assess the pharmacokinetic profiles of the RNAi agents AD-45163,AD-51544, AD-51545, AD-51546, and AD-51547, these agents, in PBS buffer,were administered to C57BL/6 mice using a single IV bolus orsubcutaneous (SC) administration. The plasma concentrations and liverconcentrations of the agents were assessed at various timepoints afterthe administration.

The plasma pharmacokinetic parameters are presented in Tables 3 and 4below. The mean resident time (MRT) in plasma was about 0.2 hours afterIV dosing and about 1 hour after SC dosing. At a dose of 25 mg/kg, theagents AD-51544, AD-51545, AD-51546, and AD-51547 showed similar plasmapharmacokinetic properties. Each of these agents had more than 75%bioavailability from the subcutaneous space. Their bioavailability wassuperior to that of the parent agent AD-45163 that was administered at ahigher dose of 30 mg/kg. The subcutaneous bioavailability of AD-51544and AD-51547 was about 100%, whereas that of AD-51545 was 90% and thatof and AD-51546 was 76%.

TABLE 3 Summary of Plasma PK Parameter Estimates After SC Administrationof TTR-GalNAc siRNAs in Mice 30 mpk 25 mpk 25 mpk 25 mpk 25 mpk AD- AD-AD- AD- AD- 45163 51544 51545 51546 51547 (h/c (h/c (h/c (h/c (h/c TTR-TTR- TTR- TTR- TTR- Parameter GalNAc) GalNAc) GalNAc) GalNAc) GalNAc)Plasma 0.25 1 0.5 1 0.5 Tmax (h) Plasma 9.6 11.7 10.9 11.7 12.1 Cmax(μg/mL) Plasma 12.4 21.9 19.9 20.9 25.3 AUC (h*μg/mL) F_(sc)(%) 79 10090.1 76.0 99.2

TABLE 4 Plasma siRNA PK Parameters in Mice after an IV Bolus or SC Doseof AD-51544, 51545, 51546 or 51547 at 25 mg/kg Test Article AD-51544AD-51545 AD-51546 AD-51547 siRNA Dose (mg/kg) 25 25 25 25 Route ofAdministration IV SC IV SC IV SC IV SC t_(max) (h) 0.083 1 0.083 0.50.083 1 0.083 0.5 C_(max) (μg/mL) 96.5^(a) 11.7 108^(a)    10.9128^(a)    10.9 123^(a)    12.1 AUC_(0-last) (h · μg/mL) 21.6 21.922.1   19.9 27.5   20.9 25.5   25.3 MRT_(0-last) (h) 0.17 1.2 0.16  1.10.22  1.4 0.19  1.3 Apparent t_(1/2 β) (h)^(b) ND ND ND 0.49 ND 1.2 ND0.56 F_(SC) (%)^(c) — 102 — 90.1 — 76.0 — 99.2 ^(a)Concentration at the1^(st) sampling time (5 min) after IV dosing ^(b)Apparent eliminationhalf-life (t_(1/2 β)) could not be determined (ND) for all 4 testarticles after IV dosing as the terminal phase of the concentration-timeprofiles was not well defined, as a result, the t_(1/2 β) -associated PKparameters (eg, AUC_(0-∞), CL and Vss) were not reported. ^(c)SCbioavailability, calculated as percentage ratio of AUC_(0-last) after SCand IV dosing at 25 mg/kg

The results also indicated that the RNAi agents AD-45163, AD-51544,AD-51545, AD-51546, and AD-51547 achieved similar or higherconcentrations in the liver when administered subcutaneously than whenadministered by IV bolus. The liver pharmacokinetic parameters arepresented in Tables 5 and 6 below. The peak concentration (C_(max)) andarea under the curve (AUC_(0-last)) in the liver were two to three timeshigher after subcutaneous administration as compared with IVadministration of the same agent at the same dose. Liver exposures werehighest for AD-51547 and lowest for AD-51545. The mean resident time(MRT) and elimination half-life were longer for AD-51546 and AD-51547compared with AD-51544 and AD-51545. Following subcutaneousadministration, the approximate MRTs were 40 hours for AD-51546 and 25hours for AD-51547, whereas the MRTs for AD-51544 and AD-51545 werelower (about 6-9 hours). The elimination half life of AD-51546 andAD-51547 was also higher (41-53 hours) than was the elimination halflife of AD-51544 and AD-51545 (6-10 hours).

TABLE 5 Summary of Liver PK Parameter Estimates After SC Administrationof TTR-GalNAc siRNAs in Mice 30 mpk 25 mpk 25 mpk 25 mpk 25 mpk AD- AD-AD- AD- AD- 45163 51544 51545 51546 51547 (h/c (h/c (h/c (h/c (h/c TTR-TTR- TTR- TTR- TTR- Parameter GalNAc) GalNAc) GalNAc) GalNAc) GalNAc)Liver Tmax 8 4 4 2 8 (h) Liver Cmax 313 126 80 117 174 (μg/g) Liver AUC4519 1092 763 2131 4583 (h*μg/g)

TABLE 6 Liver siRNA PK Parameters in Mice after an IV Bolus or SC Doseof AD-51544, 51545, 51546 or 51547 at 25 mg/kg Test Article AD-51544AD-51545 AD-51546 AD-51547 siRNA Dose (mg/kg) 25 25 25 25 Route ofAdministration IV SC IV SC IV SC IV SC t_(max) (h) 1 4 1 4 4 2 2 8C_(max) (μg/g) 67.9 126 37.0 80.5 35.3 117 73.8 174 AUC_(0-last) (h ·μg/g) 632 1092 324 763 984 2131 1429 4583 MRT_(0-last) (h) 8.7 6.5 5.98.5 45.7 40.2 29.4 25.3 Apparent t_(1/2β) (h) 8.1 8.2 5.7 10.0 51.1 45.341.1 52.7

Example 8: In Vitro Stability of RNAi Agents in Monkey Serum

The serum stability of RNAi agents AD-51544, AD-51545, AD-51546, andAD-51547 was also assessed in monkeys. The results demonstrated that theantisense and sense strands of AD-51544, AD-51545, and AD-51547 showedserum stability over a period of about 24 hours (data not shown).

Example 9: RNAi Agents Produce Lasting Suppression of TTR Protein inNon-Human Primates

The RNA silencing activity of RNAi agents AD-45163, AD-51544, AD-51545,AD-51546, and AD-51547 was assessed by measuring suppression of TTRprotein in serum of cynomologous monkeys following subcutaneousadministration of five 5 mg/kg doses (one dose each day for 5 days) or asingle 25 mg/kg dose. Pre-dose TTR protein levels in serum were assessedby averaging the levels at 11 days prior to the first dose, 7 days priorto the first dose, and 1 day prior to the first dose. Post-dose serumlevels of TTR protein were assessed by determining the level in serumbeginning at 1 day after the final dose (i.e., study day 5 in the 5×5mg/kg group and study day 1 in the 1×25 mg/kg group) until 49 days afterthe last dose (i.e., study day 53 in the 5×5 mg/kg group and study day49 in the 1×25 mg/kg group). See FIG. 13 .

TTR protein levels were assessed as described in Example 6. The resultsare shown in FIGS. 14A and 14B and in Tables 7 and 8.

A maximal suppression of TTR protein of up to about 50% was achieved inthe groups that received 25 mg/kg of AD-45163, AD-51544, AD-51546, andAD-51547 (see Table 8). A greater maximal suppression of TTR protein ofabout 70% was achieved in the groups that received 5×5 mg/kg ofAD-45163, AD-51544, AD-51546, and AD-51547 (see Table 7). The agentAD-51545 produced a lesser degree of suppression in both administrationprotocols. Significant suppression of about 20% or more persisted for upto 49 days after the last dose of AD-51546 and AD-51547 in both the 1×25mg/kg and 5×5 mg/kg protocols. Generally, better suppression wasachieved in the 5×5 mg/kg protocol than in the 1×25 mg/kg protocol.

TABLE 7 Fraction Serum Transthyretin Relative to Pre-dose in CynomolgusMonkeys (5 mg/kg daily for 5 days) D-11 D-7 D-1 D5 D7 D9 D11 D14 D18 D22D26 D32 D39 D46 D53 AD-45163 0.98 0.99 1.03 0.71 0.52 0.40 0.34 0.270.31 0.39 0.48 0.64 0.68 0.81 0.88 AD-51544 1.02 0.99 0.99 0.60 0.470.37 0.35 0.39 0.48 0.58 0.66 0.74 0.83 0.91 0.92 AD-51545 1.03 0.971.00 0.73 0.65 0.63 0.69 0.68 0.78 0.87 0.97 1.00 1.03 1.06 1.09AD-51546 1.01 0.97 1.02 0.59 0.42 0.35 0.30 0.32 0.43 0.58 0.66 0.770.92 0.93 0.97 AD-51547 0.99 0.99 1.02 0.74 0.54 0.41 0.34 0.34 0.390.49 0.51 0.53 0.65 0.70 0.77

TABLE 8 Fraction Serum Transthyretin Relative to Pre-dose in CynomolgusMonkeys (25 mg/kg) D-11 D-7 D-1 D1 D3 D5 D7 D10 D14 D18 D22 D28 D35 D42D49 AD-45163 1.04 1.01 0.95 0.99 0.84 0.67 0.57 0.44 0.45 0.51 0.58 0.660.72 0.78 0.85 AD-51544 1.01 1.04 0.95 0.92 0.69 0.57 0.49 0.48 0.560.65 0.69 0.77 0.83 0.87 0.94 AD-51545 0.98 1.02 0.99 0.87 0.77 0.690.71 0.72 0.84 0.90 0.92 0.99 1.00 1.00 1.00 AD-51546 1.04 1.03 0.930.89 0.71 0.62 0.53 0.50 0.55 0.70 0.70 0.69 0.72 0.79 0.84 AD-515470.96 1.03 1.01 1.19 0.90 0.70 0.54 0.48 0.50 0.50 0.52 0.58 0.62 0.700.72

Example 10: Tolerability of RNAi Agents that Target TTR In CytokineEvaluation in Whole Blood Assay

To assess the tolerability of RNAi agents that target TTR (includingAD-45163, AD-51544, AD-51545, AD-51546, and AD-51547), each agent wastested in a whole blood assay using blood from three human donors. Theagents were either 300 nM DOTAP transfected or 1 μM without transfectionreagent (free siRNA). There was less than a two fold change for thefollowing cytokines/chemokines: G-CSF, IFN-γ, IL-10, IL-12 (p70), IL1β,IL-Ira, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-M, TNFα. (Results notshown).

In Vivo Evaluation

To assess in vivo tolerability, RNAi agents were injected subcutaneouslyin CD1 mice at a dose of 125 mg/kg. No cytokine induction was observedat 2, 4, 6, 24, or 48 hours after subcutaneous injection of AD-45163. Nosignificant cytokine induction was observed at 6 or 24 hours aftersubcutaneous injection of AD-51544, AD-51545, AD-51546, or AD-51547.

To further assess in vivo tolerability, multiple RNAi agents (includingAD-45163, AD-51544, AD-51545, AD-51546, and AD-51547) were tested bysubcutaneous injection of 5 and 25 mg in non-human primates(cynomologous monkeys) with dose volumes between 1-2 ml per site. Noerythema or edema was observed at injection sites.

Single SC Dose Rat Tolerability Study

To assess toxicity, rats were injected with a single subcutaneous doseof 100, 250, 500, or 750 mg/kg of AD-45163 (see Table 9). The followingassessments were made: clinical signs of toxicity, body weight,hematology, clinical chemistry and coagulation, organ weights (liver &spleen); gross and microscopic evaluation (kidney, liver, lung, lymphnode, spleen, testes, thymus, aorta, heart, intestine (small and large).

TABLE 9 Single SC Dose Rat Tolerability Study: 100, 250, 500 & 750 mg/kgof AD-45163 in Sprague Pawley Rats Dose Dose No. Male Level Volume Route& Sprague Day of Group (mg/kg) (ml/kg) Regimen Dawley Rats Necropsy PBS0 10 SC 7/group Day 4 AD-45163 100 Injection (5 Tox Parent Day 1animals, (2 sites) 2 TK animals)

The results showed no test article-related clinical signs of toxicity,effects on body weight, organ weights, or clinical chemistry. Nohistopathology was observed in heart, kidneys, testes, spleen, liver,and thymus. There was a non-adverse, slight test article-relatedincrease in WBC (Q68%, primarily attributed to increase in NEUT andMONO) at 750 mg/kg. These results indicate that a single-dose of up to750 mg/kg is well tolerated in rats.

Tolerability of Repeated Subcutaneous Administrations in Rats

To assess the tolerability of repeated subcutaneous administrations ofAD-45163, daily subcutaneous injections of 300 mg/kg were given for 5days, and a necropsy was performed on day 6. The study design is shownin Table 10.

TABLE 10 Five Day Repeat Dose Tolerability Study in Rat Dose Level ConcNo of Tox Group (kmg/kg (mg/mL) Animals Nx Day 6 PBS 0 0 2M, 2F 2M, 2FAD-45163 300 150 2M, 2F 2M, 2F

The following outcome variables were assessed: clinical signs, bodyweights, hematology, clinical chemistry and coagulation, organ weights,gross and microscopic evaluation (liver, spleen, kidney, heart, GI tractand first and last injection site). The results showed no testarticle-related clinical signs, body weight or organ weight effects, andalso no test article-related findings in clinical hematology orchemistry. There was a possible slight prolongation of activated partialthromboplastin time (APTT) on day 6 (20.4 vs. 17.4 sec). Histopathologyrevealed no test article-related findings in the liver, spleen, heart,and GI tract. In the kidney, minimal to slight hypertrophy of thetubular epithelium (not adverse) was observed. At the last injectionsite, there was minimal multifocal mononuclear infiltration, notadverse. These results indicate that five daily 300 mg/kg doses of theparent RNAi agent AD-45163 are well tolerated in rats.

Example 11: RNAi Agents Produce Lasting Suppression of TTR Protein inNon-Human Primates

The RNA silencing activity of RNAi agent AD-51547 was assessed bymeasuring suppression of TTR protein in the serum of cynomologousmonkeys following subcutaneous administration of a “loading phase” ofthe RNAi agent: five daily doses of either 2.5 mg/kg, 5 mg/kg or 10mg/kg (one dose each day for 5 days) followed by a “maintenance phase”of the RNAi agent: weekly dosing of either 2.5 mg/kg, 5 mg/kg or 10mg/kg for 4 weeks. Pre-dose TTR protein levels in serum were assessed byaveraging the levels at 11 days prior to the first dose, 7 days prior tothe first dose, and 1 day prior to the first dose. Post-dose serumlevels of TTR protein were assessed by determining the level in serumrelative to pre-dose beginning at 1 day after the loading phase wascompleted until 40 days after the last dose of the maintenance phase(i.e., study day 70).

TTR protein levels were assessed as described in Example 6. The resultsare shown in FIG. 15 .

A maximal suppression of TTR protein of up to about 80% was achieved inall of the groups that received either 2.5 mg/kg, 5 mg/kg or 10 mg/kg ofAD-51547. Nadir knockdown was achieved in all of the groups by about day14, the suppression sustained at nadir knockdown levels with a weeklymaintenance dose of either 2.5 mg/kg, 5 mg/kg or 10 mg/kg of AD-51547.The levels of TTR had not returned to baseline more than 40 days afterthe day of administration of the last maintenance dose for the 5 and 2.5mg/kg dose levels.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. A double stranded RNAi agent comprising a sense strand complementaryto an antisense strand, wherein said antisense strand comprises a regioncomplementary to part of an mRNA encoding transthyretin (TTR), whereineach strand ja independently 15 to 30 nucleotides in length, whereinsaid double stranded RNAi agent is represented by formula (III):sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′   (III) wherein: i, j, k, and l are each independently 0 or 1;p, p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′independently represents an oligonucleotide sequence comprising 0-25nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof, each n_(p), n_(p)′,n_(q), and n_(q)′ independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and whereinthe sense strand is conjugated to at least one ligand.
 2. The RNAi agentof claim 1, wherein i is 1; j is 1; or both i and j are 1; or wherein kis 1; l is 1; or both k and l are
 1. 3. (canceled)
 4. The RNAi agent ofclaim 1, wherein XXX is complementary to X′X′X′, YYY is complementary toY′Y′Y′, and ZZZ is complementary to Z′Z′Z′. 5.-10. (canceled)
 11. TheRNAi agent of claim 1, wherein the duplex region is 15-30 nucleotidepairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotidepairs in length; 23-27 nucleotide pairs in length; 19-21 nucleotidepairs in length; or 21-23 nucleotide pairs in length. 12.-17. (canceled)18. The RNAi agent of claim 1, wherein the modifications on thenucleotides are selected from the group consisting of LNA, HNA, CeNA,2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro,2′-deoxy, 2′-hydroxyl, and combinations thereof.
 19. The RNAi agent ofclaim 18, wherein the modifications on the nucleotides are 2′-O-methyl,2′-fluoro or both.
 20. The RNAi agent of claim 1, wherein the ligand isone or more GalNAc derivatives attached through a bivalent or trivalentbranched linker.
 21. The RNAi agent of claim 1, wherein the ligand is

and wherein the ligand is attached to the 3′ end of the sense strand.22. (canceled)
 23. (canceled)
 24. The RNAi agent of claim 1 furthercomprising at least one phosphorothioate or methylphosphonateinternucleotide linkage. 25.-41. (canceled)
 42. A pharmaceuticalcomposition comprising the RNAi agent of claim
 1. 43.-49. (canceled) 50.A method of inhibiting expression of a transthyretin (TTR) in a cellcomprising contacting said cell with the RNAi agent of claim 1 or withthe pharmaceutical composition of claim 42 in an amount effective toinhibit expression of said TTR in said cell, thereby inhibitingexpression of said transthyretin (TTR) in said cell.
 51. (canceled) 52.(canceled)
 53. The method of claim 50, wherein said cell is presentwithin a subject.
 54. The method of claim 53, wherein said subject is ahuman. 55.-78. (canceled)
 79. A method of treating or preventing aTTR-associated disease in a subject, comprising administering to saidsubject a therapeutically effective amount or a prophylacticallyeffective amount of the RNAi agent of claim 1 or the pharmaceuticalcomposition of claim 42, thereby treating or preventing saidTTR-associated disease in said subject.
 80. (canceled)
 81. The method ofclaim 79, wherein said subject is a human.
 82. The method of claim 79,wherein said subject is a subject suffering from a TTR-associateddisease.
 83. (canceled)
 84. The method of claim 79, wherein said subjectcarries a TTR gene mutation that is associated with the development of aTTR-associated disease.
 85. The method of claim 79, wherein saidTTR-associated disease is selected from the group consisting of senilesystemic amyloidosis (SSA), systemic familial amyloidosis, familialamyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy(FAC), leptomeningeal/Central Nervous System (CNS) amyloidosis, andhyperthyroxinemia.
 86. (canceled)
 87. (canceled)
 88. The method of claim79, wherein said RNAi agent is administered to said subject viasubcutaneous, intramuscular or intravenous administration. 89.-96.(canceled)
 97. The method of claim 79, further comprising assessing thelevel of TTR mRNA expression or TTR protein expression in a samplederived from the subject. 98.-113. (canceled)